U.S. patent application number 11/884287 was filed with the patent office on 2009-08-13 for surface-located streptococcus pneumoniae polypeptides.
Invention is credited to Anders Boysen, Thomas Kofoed, Christian Morsczeck, Pia Nyborg Nielsen, Jorgen Petersen, Tatyana A. Prokhorova, Petra Schrotz-King, Jens Sigh.
Application Number | 20090202528 11/884287 |
Document ID | / |
Family ID | 36588972 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090202528 |
Kind Code |
A1 |
Kofoed; Thomas ; et
al. |
August 13, 2009 |
Surface-Located Streptococcus Pneumoniae Polypeptides
Abstract
The present invention relates to cell-surface-located
polypeptides of Streptococcus pneumoniae and their use in
immunisation against Streptococcal infection, in diagnosis of
Streptococcus and in identification of compounds with
anti-Streptococcus activity. In a further aspect, the invention
relates to antibodies capable of recognising cell surface-located
polypeptides of Streptococcus pneumoniae and uses thereof.
Inventors: |
Kofoed; Thomas; (Odense,
DK) ; Nyborg Nielsen; Pia; (Odense, DK) ;
Schrotz-King; Petra; (Odense, DK) ; Petersen;
Jorgen; (Odense, DK) ; Boysen; Anders;
(Odense, DK) ; Prokhorova; Tatyana A.; (Ejby,
DK) ; Sigh; Jens; (Frederiksberg, DK) ;
Morsczeck; Christian; (Odense, DK) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE LLP - San Francisco
505 MONTGOMERY STREET, SUITE 800
SAN FRANCISCO
CA
94111
US
|
Family ID: |
36588972 |
Appl. No.: |
11/884287 |
Filed: |
February 9, 2006 |
PCT Filed: |
February 9, 2006 |
PCT NO: |
PCT/DK06/00073 |
371 Date: |
June 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60653932 |
Feb 18, 2005 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/139.1; 424/190.1; 424/199.1; 424/200.1; 424/93.2; 435/235.1;
435/252.33; 435/32; 435/320.1; 435/7.34; 436/501; 514/1.1; 530/350;
530/387.1; 530/387.3; 530/387.9; 530/388.4; 536/23.1 |
Current CPC
Class: |
G01N 33/56944 20130101;
Y02A 50/482 20180101; Y02A 50/30 20180101; A61P 31/04 20180101;
A61P 37/04 20180101; C07K 14/3156 20130101 |
Class at
Publication: |
424/133.1 ;
530/387.1; 530/350; 536/23.1; 435/320.1; 435/235.1; 435/252.33;
514/12; 424/190.1; 530/388.4; 530/387.3; 424/139.1; 424/93.2;
530/387.9; 424/199.1; 424/200.1; 435/7.34; 436/501; 435/32 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C07K 14/00 20060101
C07K014/00; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 7/00 20060101 C12N007/00; C12N 1/21 20060101
C12N001/21; A61K 38/16 20060101 A61K038/16; A61K 39/09 20060101
A61K039/09; A61K 35/76 20060101 A61K035/76; A61K 31/7088 20060101
A61K031/7088; G01N 33/569 20060101 G01N033/569; G01N 33/566
20060101 G01N033/566; C12Q 1/18 20060101 C12Q001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 11, 2005 |
DK |
PA 2005 00207 |
Aug 29, 2005 |
DK |
PA 2005 01194 |
Oct 18, 2005 |
PA |
PA 2005 01463 |
Claims
1. A composition for use as a medicament, comprising one of the
group consisting of: a) an antibody capable of binding a
polypeptide selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ
ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID
NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ
ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,
SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ
ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77,
SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID
NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ
ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,
SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID
NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ
ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID
NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,
SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID
NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID
NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,
SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,
SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID
NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146,
SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID
NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155,
SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID
NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164,
SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID
NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID
NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182,
SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID
NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191,
SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID
NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200,
SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID
NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209,
SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID
NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218,
SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID
NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227,
SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID
NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236,
SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID
NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245,
SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID
NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254,
SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID
NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263,
SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID
NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272,
SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID
NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281,
and SEQ ID NO:282, b) a polypeptide selected from the group
consisting of the polypeptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID
NO:282. c) an antigenic fragment of said polypeptide, d) a variant
of said polypeptide. e) a polynucleotide comprising a sequence
encoding said polypeptide, f) an expression vector comprising a
sequence encoding said polypeptide, and g) a recombinant virus or
recombinant cell comprising said polynucleotide or said expression
vector.
2. The composition of claim 1, wherein a minimum sequence identity
of said variant of said polypeptide with said sequence is selected
from the group consisting of 95%, 96%, 97% 98%, and 99%.
3. The composition of claim 1, wherein a percentage of the full
length of said sequence contained in said antigenic fragment is
less than a value selected from the group consisting of 99%, 75%,
50%, 25%, 20%, 15%, and 10%.
4. The composition of claim 1, wherein the number of consecutive
amino acid residues of said sequence contained within said
antigenic fragment is less than a number selected from the group
consisting of 70, 50, 40, 30, and 20.
5. The composition of claim 1, wherein the minimum number of
consecutive amino acids of said sequence contained within said
antigenic fragment is selected from the group consisting of 6, 7,
8, 9, and 10.
6. The composition of claim 1, wherein the composition comprises at
least two polypeptides selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:110, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ
ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,
SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID
NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ
ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62,
SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID
NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ
ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,
SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID
NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ
ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,
SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID
NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ
ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108,
SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID
NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118,
SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127,
SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID
NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136,
SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID
NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145,
SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID
NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154,
SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID
NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163,
SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID
NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172,
SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID
NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181,
SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID
NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190,
SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID
NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199,
SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID
NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,
SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID
NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217,
SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID
NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226,
SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID
NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235,
SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID
NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244,
SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID
NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253,
SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID
NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262,
SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID
NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271,
SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275, SEQ ID
NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280,
SEQ ID NO:281, and SEQ ID NO:282.
7-13. (canceled)
14. The composition of claim 1, wherein said polypeptide comprises
an affinity tag.
15. The composition of claim 1, wherein said recombinant cell is an
attenuated or reduced-virulence Escherichia coli cell or an
attenuated or reduced-virulence Salmonella cell.
16. The composition of claim 1, wherein said recombinant cell is
alive.
17. The composition of claim 1, wherein said recombinant cell is
dead.
18. The composition of claim 1, wherein the medicament is a
vaccine.
19. The composition of claim 1, further comprising an immunogenic
carrier, wherein the immunogenic carrier is bound to said
polypeptide.
20. The composition of claim 1, further comprising an adjuvant.
21. The composition of claim 1, wherein said antibody is capable of
binding an intact Streptococcus pneumoniae cell.
22. The composition of claim 1, wherein said antibody is
polyclonal.
23. The composition of claim 1, wherein said antibody is
monoclonal.
24. The composition of claim 1, wherein said antibody is a human
antibody or humanized antibody.
25. The composition of claim 1, wherein the antibody is an
antigen-binding fragment of an antibody.
26. The composition of claim 1, wherein the antibody has a
dissociation constant Kd of less than a value selected from the
group consisting of 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-8M, 10.sup.-8M,
5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M, 10.sup.-10M,
5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M, 10.sup.-12M,
5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14Mg,
5.times.10.sup.-15M, 10.sup.-15M.
27. The composition of claim 1, wherein said composition
additionally comprises a pharmaceutically-acceptable carrier.
28. The composition of claim 1, wherein the composition is suitable
for systemic administration.
29. The composition of claim 1, wherein said composition is
suitable for a mode of administration selected from the group
consisting of intravenous, intramuscular, and subcutaneous
administration.
30. The composition of claim 1, wherein said composition is
suitable for oral administration.
31. The composition of claim 1, wherein said composition is
suitable for intranasal administration.
32. An antibody capable of binding a polypeptide having an amino
acid sequence selected from the group consisting of preferably
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID
NO:282.
33. The antibody of claim 32, wherein said antibody is capable of
binding an intact Streptococcus pneumoniae cell.
34. (canceled)
35. A recombinant cell transformed or transfected with a
polynucleotide comprising a sequence encoding a polypeptide, said
polypeptide comprising one of the group consisting of: a) a
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ
ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID
NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ
ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63,
SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ
ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77,
SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID
NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ
ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,
SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID
NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ
ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID
NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109,
SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID
NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID
NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,
SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,
SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID
NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146,
SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID
NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155,
SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID
NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164,
SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID
NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173,
SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID
NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182,
SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID
NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191,
SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID
NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200,
SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID
NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209,
SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID
NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218,
SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID
NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227,
SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID
NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236,
SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID
NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245,
SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID
NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254,
SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID
NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263,
SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID
NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272,
SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID
NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281,
and SEQ ID NO:282: b) an antigenic fragment of said sequence; and
c) a variant of said sequence.
36. The recombinant cell of claim 35, wherein the recombinant host
cell is selected from the group consisting of an Escherichia coli
cell and a Salmonella cell.
37. The recombinant cell of claim 35, wherein said recombinant cell
is an attenuated or reduced-virulence cell.
38. A method of immunizing an animal against bacterial infections,
comprising administering to the animal a composition comprising a
component selected from the group consisting of: a) a polypeptide
which comprises a sequence selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID
NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ
ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID
NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID
NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ
ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80,
SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID
NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ
ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,
SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103,
SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113,
SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,
SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID
NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131,
SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID
NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,
SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID
NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149,
SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID
NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158,
SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID
NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167,
SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID
NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176,
SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID
NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185,
SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID
NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,
SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID
NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203,
SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID
NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212,
SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID
NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221,
SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID
NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230,
SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID
NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239,
SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID
NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248,
SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID
NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257,
SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID
NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266,
SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID
NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275,
SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID
NO:280, SEQ ID NO:281, and SEQ ID NO:282, b) an antigenic fragment
of said sequence, c) a variant of said sequence, d) a
polynucleotide comprising a sequence encoding said polypeptide, e)
an expression vector comprising a sequence encoding said
polypeptide, and f) a recombinant virus or recombinant cell
comprising said polynucleotide or said expression vector.
39. The method of claim 38, wherein administration of said
composition induces a protective immune response.
40. The method of claim 38, wherein the composition is suitable for
parenteral, intravenous, intramuscular, subcutaneous, oral or
intranasal administration.
41-45. (canceled)
46. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal an antibody
capable of binding a polypeptide selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID
NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ
ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID
NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID
NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ
ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80,
SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID
NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ
ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,
SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103,
SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113,
SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,
SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID
NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131,
SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID
NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140,
SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID
NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149,
SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID
NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158,
SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID
NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167,
SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID
NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176,
SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID
NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185,
SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID
NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194,
SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID
NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203,
SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID
NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212,
SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID
NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221,
SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID
NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230,
SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID
NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239,
SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID
NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248,
SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID
NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257,
SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261, SEQ ID
NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266,
SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID
NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:275,
SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279, SEQ ID
NO:280, SEQ ID NO:281, and SEQ ID NO:282.
47. (canceled)
48. A method for raising antibodies to a polypeptide selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,
SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID
NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ
ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,
SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID
NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ
ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74,
SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ
ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88,
SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ
ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106,
SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID
NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116,
SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID
NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125,
SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID
NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134,
SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID
NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143,
SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID
NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152,
SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID
NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161,
SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID
NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170,
SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID
NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179,
SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID
NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188,
SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID
NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197,
SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID
NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206,
SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID
NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215,
SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID
NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224,
SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID
NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233,
SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID
NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242,
SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID
NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251,
SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID
NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260,
SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID
NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269,
SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID
NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278,
SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID NO:282 in a
non-human animal, comprising the steps of a. introducing into said
animal a composition comprising one of the group consisting of: i)
said polypeptide, ii) an antigenic fragment of said sequence, iii)
a variant of said sequence, iv) a polynucleotide comprising a
sequence encoding said polypeptide, v) an expression vector
comprising a sequence encoding said polypeptide, and vi) a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector; b. raising antibodies in
said animal, and c. isolating the antibodies.
49-50. (canceled)
51. A method for detecting Streptococcus pneumoniae or cellular
fragments thereof in a sample, comprising the steps of a. providing
an indicator moiety capable of specifically binding a polypeptide
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69 SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID
NO:282, b. contacting said sample with said indicator moiety; and
c. determining whether a signal has been generated by the indicator
moiety, thereby detecting whether said sample contains
Streptococcus pneumoniae or cellular fragments thereof.
52. The method of claim 51, wherein the indicator moiety is capable
of binding an intact Streptococcus pneumoniae cell.
53. The method of claim 51, wherein said indicator moiety does not
pass through the membrane of a Streptococcus pneumoniae cell.
54. The method of claim 51 to 53, wherein said indicator moiety
comprises an antibody.
55. A method for detecting Streptococcus pneumoniae or cellular
fragments thereof in a sample comprising the step of analyzing said
sample by mass spectrometry to detect the presence of a polypeptide
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID
NO:282.
56. A method for identifying a binding partner of a polypeptide
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282,
and a fragment of any of SEQ ID NOS: 1-282, comprising the steps of
a. providing said polypeptide; b. contacting said polypeptide with
a putative binding partner, and c. determining whether said
putative binding partner is capable of binding to said polypeptide
or fragment.
57. A method for identifying a compound with antibacterial activity
against Streptococcus pneumoniae, comprising the steps of a.
providing a sensitized cell having a reduced level of a polypeptide
selected from the group consisting of SEQ ID NO:1-282, preferably
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID
NO:282, and b. determining the sensitivity of said cell to a
putative antibacterial compound.
58. A method for identifying an inhibitor of a polypeptide selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:10,
SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID
NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120,
SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID
NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,
SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147,
SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID
NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165,
SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,
SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID
NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183,
SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID
NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID
NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:21,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219,
SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID
NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,
SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID
NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,
SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID
NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246,
SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID
NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255,
SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID
NO:260, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264,
SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273,
SEQ ID NO:274, SEQ ID NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID
NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, and SEQ ID
NO:282, comprising the steps of a. providing two cells that differ
in the level of said polypeptide b. determining the sensitivity of
said cells to a putative inhibitor; and c. ascertaining whether
said two cells are differently affected by the presence of said
putative inhibitor.
59. The method of claim 58, wherein the putative inhibitor does not
pass through the membrane of a Streptococcus pneumoniae cell.
60. The method of claim 58, wherein the determining step comprises
a growth assay.
61. The antibody of claim 32, wherein said antibody is
polyclonal.
62. The antibody of claim 32, wherein said antibody is
monoclonal.
63. The antibody of claim 32, wherein said antibody is a human
antibody or humanized antibody.
64. The antibody of claim 32, wherein said antibody is an
antigen-binding fragment of an antibody.
65. The antibody of claim 32, wherein the antibody has a
dissociation constant Kd of less than a value selected from the
group consisting of 5.times.10.sup.-6M, 10.sup.-16M,
5.times.10.sup.-17M, 10.sup.-17M, 5.times.10.sup.-8M, 10.sup.-8M,
5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M, 10.sup.-10M,
5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M, 10.sup.-12M,
5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M, 10.sup.-14M,
5.times.10.sup.-15M, and 10.sup.-15M.
66. The method of claim 38, wherein the animal is a human.
67. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal the antibody of
claim 33.
68. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal the antibody of
claim 61.
69. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal the antibody of
claim 62.
70. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal the antibody of
claim 63.
71. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal the antibody of
claim 64.
72. A method for treating or preventing Streptococcus infections in
an animal, comprising administering to said animal the antibody of
claim 65.
73. The method of claim 48, additionally comprising purifying said
antibodies.
74. The method of claim 48, further comprising selecting antibodies
capable of binding an intact Streptococcus pneumoniae cell.
75. The method of claim 48, wherein the animal is a transgenic
animal capable of producing human antibodies.
76. The method of claim 57, wherein the determining step comprises
a growth assay.
Description
[0001] All patent and non-patent references cited in this
application are hereby incorporated by reference in their entirety.
This patent application claims the benefit of priority from U.S.
Provisional Application Ser. No. 60/653,932, filed Feb. 18, 2005,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to cell-surface-located
polypeptides of Streptococcus pneumoniae and their use in
immunisation against Streptococcal infection, in diagnosis of
Streptococcus and in identification of compounds with
anti-Streptococcus activity.
BACKGROUND OF THE INVENTION
Occurrence of Streptococcus Infections
[0003] Sternberg and Pasteur were the first to identify
Streptococcus pneumoniae, initially described as the pneumococcus
(Austrian R. The pneumococcus at the millennium: not down, not out.
J Infect Dis 1999; 179 (Suppl 2):S338-41). Streptococcus pneumoniae
is a Gram-positive encapsulated coccus. Based on differences in the
composition of the polysaccharide capsule, about 90 serotypes are
identified. This capsule is an essential virulence factor. The
majority of pneumococcal disease in infants is associated with a
small number of these serotypes, which may vary by region. Current
data suggest that the 11 most common serotypes cause at least 75%
of invasive disease in all regions.
[0004] Streptococcus pneumoniae is a human pathogen. The reservoir
for pneumococci is presumably the nasopharynx of asymptomatic human
carriers. There is no animal or insect vector. Streptococcus
pneumoniae is the most common cause of bacteremia, pneumonia,
meningitis and otitis media in young children. Pneumococcal disease
is a very serious illness in young children. In the United States
it is estimated that Streptococcus pneumoniae cause 200 deaths, 700
cases of meningitis, 17,000 cases of bacteremia, 4.9 million cases
of otitis media (ear infections) annually in children under 5 years
of age. In Europe and the United States, pneumococcal pneumonia is
the most common community-acquired bacterial pneumonia, estimated
to affect approximately 100 per 100 000 adults each year. The
corresponding figures for febrile bacteremia and meningitis are
15-19 per 100,000 and 1-2 per 100,000, respectively. The risk for
one or more of these manifestations is much higher in infants and
elderly people.
[0005] Meningitis is the most severe type of pneumococcal disease.
Of children under 5 years with pneumococcal meningitis, about 5%
will die of their infection and others may have long-term problems
such as hearing loss. Many children with pneumococcal pneumonia or
blood stream infections will be ill enough to be hospitalized;
about 1% of children with blood stream infections or pneumonia with
a blood stream Infection will die of their illness. Nearly all
children with ear infections recover, although children with
recurrent infections can suffer hearing loss.
[0006] At serious risk are also patients taking immunosuppressive
chemotherapy, those with congenital and acquired immune deficiency
(including HIV infections) and those with chronic renal disease.
Table. 1: The major disease indications and the number of
hospitalised patients as well as case fatality rates in children
and the elderly, which occur per annum in the US:
TABLE-US-00001 Pneumococcal Pneumococcal Pneumococcal Disease
indication pneumonia bacteraemia meningitis Hospitalised patients/
175.000 50.000 3.000-6.000 annum in the US Case fatality rate
5-7%/higher 20%/60% 30%/80% children/elderly
TABLE-US-00002 TABLE 2 Incidence, case-fatality ratio, projected
U.S. cases and deaths, and proportion nonsusceptible to penicillin
of invasive disease identified in the Active Bacterial Core
surveillance (ABCs), 1998 Group A Group B Haemophilus Neisseria
Streptococcus Streptococcus Streptococcus influenzae meningitidis
pneumoniae Aggregate incidence.sup.a 3.8 6.5 1.4 1.0 24.1 Rauge by
area.sup.a 2.6-4.1 4.8-8.5 1.1-2.3 0.6-2.0 20.0-28.9 Case- fatality
ratio 12.2% 9.5% 13.9% 13.7% 9.3% in ABCs areas Projected U.S.
cases 10,200 17.400 3.900 2.500 63,000 Projected U.S. deaths 1,300
1,700 500 400 6,100 Penicillin nonsusceptibility.sup.b 0 0 -- 1.1%
25.0% .sup.aIncidence = cases per 100,000. .sup.bNonsusceptible
includes isolates classified as either intermediate or resistant to
penicillin. Results reflect testing of group A streptococcal
isolates from 1997 (n = 183) and group B streptococcal isolates
from 1997 and 1998 combined (n = 188).
[0007] Schuchat, A et al. "Active Bacterial Core Surveillance of
the Emerging Infections. Program Network", Emerging Infectious
Diseases, Vol. 7, No. 1, January-February 2001.
Symptoms of Streptococcus pneumoniae Infections
[0008] Pneumococcal pneumonia is the most common clinical
presentation of pneumococcal disease among adults. The incubation
period of pneumococcal pneumonia is short, about 1 to 3 days.
Symptoms generally include an abrupt onset of fever and chills or
rigors. Typically there is a single rigor, and repeated shaking
chills are uncommon. Other common symptoms include pleuritic chest
pain, cough productive of mucopurulent, rusty sputum, dyspnea
(shortness of breath), tachypnea (rapid breathing), hypoxia (poor
oxygenation), tachycardia (rapid heart rate), malaise, and
weakness.
Treatment and Prevention of Streptococcus pneumoniae Infections
[0009] The emerging resistance to penicillin and other commonly
used antibiotics underscores the importance of the development of
novel strategies to combat pneumococcal disease. In some areas of
the U.S. up to 40% of invasive pneumococcal isolates are resistant
to penicillin. Treatment will usually include a broad spectrum
cephalosporin, and often vancomycin, until results of antibiotic
sensitivity testing are available.
[0010] There are two vaccines against Streptococcus pneumoniae
available on the market: [0011] 1. Prevnar.RTM. (Wyeth), a 7-valent
pneumococcal conjugate vaccine, containing polysaccharides of
serotype 4, 6B, 9V, 14, 18C, 19F and 23F. [0012] 2. Pneumovax.RTM.
(Merck Research Laboratories), a 23-valent polysaccharide vaccine
containing 23 purified capsular polysaccharide antigens (serotypes
1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C,
19A, 19F, 20, 22F, 23F, and 33F).
[0013] However, there is still a large medical need for development
of improved Streptococcus vaccines, because: [0014] These vaccines
only cover certain serotypes, e.g. Prevnar.RTM. has a potential
coverage of over 85% of the pneumococcal isolates for the USA,
60-70% for Europe and around 55% for Asia. [0015] Children under 2
years of age, who suffer the highest rates of pneumococcal carriage
and disease, and immunocompromised patients show a severely
impaired antibody response upon this vaccination. [0016] The
polysaccharide vaccines are not effective against acute otitis
media caused by Streptococcus pneumoniae. [0017] The polysaccharide
vaccines do not induce a T-cell-dependent immune response. This
implicates the absence of memory B cells and limits the period of
protection. [0018] Several of the capsule polysaccharides are
poorly immunogenic. These include several serotypes associated with
penicillin resistance.
[0019] Currently, several pneumococcal surface proteins are
considered as alternative vaccine candidates because of their
serotype-independence. However, so far, none of the proteins are
considered to elicit species-wide pneumococcal protection. This can
be explained by the occurrence of allelic variation within most
individual proteins. Antibodies raised against a single protein may
not recognize allelic variants. Efficacy against pneumonia is an
important factor in deciding on the use of new vaccines in
developing countries.
[0020] In addition to better ways of treatment and prevention,
there is a need for novel rapid and reliable methods for diagnosis
of Streptococcus pneumoniae infections. The above objectives can be
accomplished through the identification and use of suitable
Streptococcus pneumoniae polypeptides that can function as targets,
i.e. targets for the immune system and/or for antibodies, targets
for cytotoxic inhibitors, or targets for indicator moieties in
diagnosis.
SUMMARY OF THE INVENTION
[0021] The present application relates to surface-located
polypeptides of Streptococcus pneumoniae. In the context of this
application, a `surface-located` polypeptide is defined as a
polypeptide which is at least partially (i.e. part of the
polypeptide chain and/or part of the population of polypeptide
molecules) localised outside the membrane of a Streptococcus
pneumoniae cell. Thus, a surface-located polypeptide is a
polypeptide which is fully or partially exposed to the space
outside the membrane. Surface-located polypeptides furthermore
include all polypeptides or polypeptide fragments that can be
identified in fractions obtained by high-pH surface-protein
extraction or mutanolysin digestion as described herein.
[0022] Surface-located polypeptides are attractive targets for
antibacterial therapy and/or diagnosis of bacterial infection,
since the exposure of such polypeptides to the extracellular space
means that compounds that interact with these polypeptides (e.g.
compounds used to prevent, treat or diagnose bacterial infections)
often do not need to enter or pass the membrane to be
effective.
[0023] The determination of cell-surface localisation of a
Streptococcus pneumoniae polypeptide can at present only be done
experimentally and not by bioinformatics, as no common sorting
signals or motifs are known for this localisation. It is possible
to predict with some degree of certainty whether or not
polypeptides enter the periplasm, but no general motif has been
identified for surface-localisation of polypeptide. Prior art
strategies for the identification of candidates for protein
vaccination against Streptococcus pneumoniae have mainly been based
on genome sequencing and in silico analysis (WO 02/077021; Wizemann
et al. (2001) Infect. Immun. 69:1593-1598). These strategies have
not been very successful, as only a small subpopulation of the
candidates identified and tested conferred protection in a mouse
model (Wizemann et al. (2001) Infect. Immun. 69:1593-1598).
[0024] The inventors have identified 282 different polypeptides in
cell-surface fractions of Streptococcus pneumoniae. The method that
was employed identifies polypeptides that are expressed at a
relatively high level. The combination of being surface-exposed and
being present in relatively high amounts makes these polypeptides
highly suitable as targets for antibodies and thus for use in
passive or active immunisation/vaccination.
[0025] Accordingly, in a first aspect, the invention relates to a
composition comprising [0026] a polypeptide which comprises a
sequence selected from the group consisting of surface-located
Streptococcus polypeptides of SEQ ID NO:1-282, or comprises an
antigenic fragment or variant of said sequence, [0027] or [0028] a
polynucleotide comprising a sequence encoding said polypeptide,
[0029] or [0030] an expression vector comprising a sequence
encoding said polypeptide, [0031] or [0032] a recombinant virus or
recombinant cell comprising said polynucleotide or said expression
vector, [0033] or [0034] an antibody capable of binding said
polypeptide, for use as a medicament.
[0035] In a preferred embodiment, said composition comprises [0036]
a polypeptide which comprises a sequence selected from the group
consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID
NO:28, or comprises an antigenic fragment or variant of said
sequence, [0037] or [0038] a polynucleotide comprising a sequence
encoding said polypeptide, [0039] or [0040] an expression vector
comprising a sequence encoding said polypeptide, [0041] or [0042] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector.
[0043] In an even more preferred embodiment, said composition
comprises [0044] a polypeptide which comprises SEQ ID NO:16, or
comprises an antigenic fragment or variant of SEQ ID NO:16, [0045]
or [0046] a polynucleotide comprising a sequence encoding said
polypeptide, [0047] or [0048] an expression vector comprising a
sequence encoding said polypeptide, [0049] or [0050] a recombinant
virus or recombinant cell comprising said polynucleotide or said
expression vector. SEQ ID NO:16 represents a homolog of
lipoate-protein ligase A, an enzyme which has previously been
identified and characterised in E. coli and L. monocytogenes
(Morris et al. (1994) J. Biol. Chem. 269:16091; O'Riordan et al.
(2003) Science 302:462). Proteins of this family have not
previously been identified on the cell surface or found to be
vaccine candidates or suitable targets for antibody therapy.
[0051] In another preferred embodiment, said composition comprises
an antibody capable of binding a polypeptide selected from the
group consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13, SEQ
ID NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:33. In an even
more preferred embodiment, said polypeptide is SEQ ID NO:16.
[0052] In a further main aspect, the invention relates to the use
of a composition comprising [0053] a polypeptide which comprises a
sequence selected from the group consisting of SEQ ID NO:1-282, or
comprises an antigenic fragment or variant of said sequence, [0054]
a polynucleotide comprising a sequence encoding said polypeptide,
[0055] an expression vector comprising a sequence encoding said
polypeptide, or [0056] a recombinant virus or recombinant cell
comprising said polynucleotide or said expression vector, for the
preparation of a medicament for the immunisation of an animal or
human being against bacteria, preferably Streptococcus, more
preferably Streptococcus pneumoniae, infections. Preferred
sequences are SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID
NO:28. Most preferred SEQ ID NO:16.
[0057] In a further main aspect, the invention relates to an
antibody capable of binding a polypeptide selected from the group
consisting of SEQ ID NO:1-282.
[0058] Furthermore, the invention relates, in a main aspect, to the
use of an antibody capable of binding a polypeptide selected from
the group consisting of SEQ ID NO:1-282 for the manufacture of a
medicament for the treatment or prevention of Streptococcus,
preferably Streptococcus pneumoniae, infections in an animal or
human being. The use of antibodies capable of binding a polypeptide
selected from the group consisting of SEQ ID NO:16, SEQ ID NO:10,
SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28 or SEQ ID
NO:33 is preferred.
[0059] Most preferred is the use of an antibody capable of binding
the polypeptide of SEQ ID NO:16 for the manufacture of a medicament
for the treatment or prevention of Streptococcus, preferably
Streptococcus pneumoniae, infections in an animal or human
being.
[0060] The combination of being surface-exposed and being present
in relatively high amounts also makes the polypeptides identified
by the inventors highly suitable as targets for diagnosis of
Streptococcus pneumoniae infection, allowing detection of intact
cells with high sensitivity. Thus, in a further main aspect, the
invention relates to methods for detecting Streptococcus pneumoniae
or parts thereof, using indicator moieties capable of recognising
the cell-surface located polypeptides described herein.
[0061] In addition, the surface-localisation of the polypeptides
makes them suitable as targets for inhibitors. Such inhibitors may
be bactericidal or bacteriostatic or prevent interaction of
Streptococcus pneumoniae with the host organism (virulence). Thus,
in a further main aspect, the invention relates to methods for
identifying inhibitors of the cell-surface located polypeptides
described herein.
DEFINITIONS
[0062] Vaccine--is used to indicate a composition capable of
inducing a protective immune response against a microorganism in a
human being or animal. [0063] Protective immune response--is used
to indicate an immune response (humoral/antibody and/or cellular)
inducing memory in an organism, resulting in the infectious agent,
herein Streptococcus pneumoniae, being met by a secondary rather
than a primary response, thus reducing its impact on the host
organism. [0064] Polypeptide--unless specified otherwise, the term
`polypeptide` when used herein can also refer to a variant or
fragment of a polypeptide. Preferred polypeptides are antigenic
polypeptides. [0065] Fragment--is used to indicate a non-full
length part of a polypeptide. Thus, a fragment is itself also a
polypeptide. [0066] Variant--a `variant` of a given reference
polypeptide refers to a polypeptide that displays a certain degree
of sequence identity to said reference polypeptide, but is not
identical to said reference polypeptide. [0067]
Antigen/antigenic/antigenicity/immunogen/immunogenic/immunogenicity
all refer to the capability of inducing an immune response. [0068]
Immunogenic carrier--refers to a compound which directly or
indirectly assists or strengthens an immune response. [0069]
Expression vector--refers to a, preferably recombinant, plasmid or
phage or virus to be used in production of a polypeptide from a
polynucleotide sequence. An expression vector comprises an
expression construct, comprising an assembly of (1) a genetic
element or elements having a regulatory role in gene expression,
for example, promoters or enhancers, (2) a structural or coding
sequence which is transcribed into mRNA and translated into
protein, and which is operably linked to the elements of (1); and
(3) appropriate transcription initiation and termination sequences.
[0070] Binding partner--of a polypeptide refers to a molecule that
can bind to said polypeptide. Such binding can be indirect, through
another molecule, but is preferably direct. A binding partner can
be any type of molecule, such as e.g. small hydrophobic molecules
or e.g. a cellular or extracellular macromolecule, such as a
protein, a carbohydrate or a nucleic acid. Preferred types of
binding partners include antibodies, ligands or inhibitors. [0071]
Plurality--the term `plurality` indicates more than one, preferably
more than 10. [0072] Indicator moiety--the term `indicator moiety`
covers a molecule or a complex of molecules that is capable of
specifically binding a given polypeptide and/or cell, and is
capable of generating a detectable signal. Preferably, the
indicator moiety is an antibody or comprises an antibody molecule.
Thus, a preferred indicator moiety is an antibody coupled to or in
complex with a detectable substance. [0073] Host-derived molecule
or host molecule--refers to a molecule which is normally found in a
host organism that can be infected with Streptococcus pneumoniae. A
host-derived molecule is preferably a host, polypeptide, preferably
a human polypeptide. [0074] Antibody--the term `antibodies` when
used herein is intended to cover antibodies as well as functional
equivalents thereof. Thus, this includes polyclonal antibodies,
monoclonal antibodies (mAbs), humanised, human or chimeric
antibodies, single-chain antibodies, and also binding fragments of
antibodies, such as, but not limited to, Fab fragments,
F(ab').sub.2 fragments, fragments produced by a Fab expression
library, anti-idiotypic antibodies, hybrids comprising antibody
fragments, and epitope-binding fragments of any of the these. The
term also includes multivalent, multispecific, such as bispecific
antibodies and mixtures of monoclonal antibodies. [0075]
Dissociation constant, Kd, is a measure to describe the strength of
binding (or affinity or avidity) between macromolecules, for
example an antibody and its antigen. The smaller Kd the stronger
binding.
[0076] Isolated--used in connection with polypeptides,
polynucleotides and antibodies disclosed herein `isolated` refers
to these having been identified and separated and/or recovered from
a component of their natural, typically cellular, environment.
Contaminant components of the natural environment are materials
that would typically interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes. Polypeptides,
polynucleotides and antibodies of the invention are preferably
isolated, and vaccines and other compositions of the invention
preferably comprise isolated polypeptides or isolated
polynucleotides or isolated antibodies.
DETAILED DESCRIPTION
Figures
[0077] FIG. 1: A table of preferred compositions of the invention.
The numbers in the columns and rows indicate SEQ ID NOs. Each cross
refers to a composition comprising the polypeptide (or antigenic
fragment or variant thereof) of the column to which the cross
belongs as well as the polypeptide (or antigenic fragment or
variant thereof) of the row to which the cross belongs.
[0078] FIG. 2: List of amino acid sequences of surface-located
Streptococcus pneumoniae polypeptides.
[0079] FIG. 3: RT-PCR with cDNA derived from a spleen from a mouse
infected with S. pneumoniae D39 at 1 day of infection. Primers were
used specific for transcripts for antigens 029 (SEQ ID NO:16), 060
(SEQ ID NO:26), 607 (SEQ ID NO:20) and 653 (SEQ ID NO:33).
Moreover, primers were used specific for transcript of the Sigma 70
subunit of the pneumococcal RNA-Polymerase (house keeping gene).
-RT: control without reverse transcriptase; +RT: RT--PCR; N:
non-template control.
[0080] FIG. 4: Immunoblot with patient serum (single patient) for
detection of rec. vac. (antigens) 029, 060, 144, 487, 607, 646 and
653.
[0081] FIG. 5: Immunogenicity of antigens (ags) 029, 060, 607, 653
and controls with untreated animals (non immunized), Alum adjuvants
alone, and an unrelated antigen at days 0, 21 and 35 of
vaccination.
[0082] FIG. 6: CFU 6 h after challenge with S. pneumoniae D39 in
blood of mice vaccinated with antigens (ags) 029, 060, 607, 653 and
controls with untreated animals (non immunized), Alum adjuvants
alone, and unrelated antigen.
[0083] FIG. 7: Survivors after challenge with S. pneumoniae D39 of
mice vaccinated with antigens (ags) 029 and 607 compared with
control groups with untreated animals (non immunized), Alum
adjuvants alone and an unrelated antigen (sigma)
COMPOSITIONS FOR USE AS A MEDICAMENT
[0084] In a first main aspect, the invention relates to a
composition comprising [0085] a polypeptide which comprises a
sequence selected from the group consisting of surface-located
Streptococcus pneumoniae polypeptides of SEQ ID NO:1-282, or
comprises an antigenic fragment or variant of said sequence, [0086]
a polynucleotide comprising a sequence encoding said polypeptide,
[0087] an expression vector comprising a sequence encoding said
polypeptide, [0088] a recombinant virus or recombinant cell
comprising said polynucleotide or said expression vector, or [0089]
an antibody capable of binding said polypeptide, for use as a
medicament.
[0090] In an important embodiment, the composition comprises [0091]
a polypeptide which comprises a sequence selected from the group
consisting of SEQ ID NO:1-282, or comprises an antigenic fragment
or variant of said sequence, [0092] a polynucleotide comprising a
sequence encoding said polypeptide, [0093] an expression vector
comprising a sequence encoding said polypeptide, or [0094] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector.
[0095] Said composition can be used as a vaccine for active
immunisation of an individual in need thereof. This is described in
the section `vaccine compositions and methods of vaccination of the
invention`.
[0096] In one preferred embodiment, the composition comprises a
polypeptide which comprises a sequence selected from the group
consisting of SEQ ID NO:1-282 or comprises antigenic fragment or
variant of said sequence.
[0097] In another important embodiment, the composition comprises
an antibody capable of binding a polypeptide selected from the
group consisting of surface-located Streptococcus pneumoniae
polypeptides of SEQ ID NO:1-282. Said composition can e.g. be used
in passive immunisation of an individual in need thereof. This is
described in the section `antibodies and methods for raising
antibodies of the invention`.
Vaccine Compositions and Methods of Vaccination of the
Invention
[0098] The goal of vaccination or active immunisation is to provide
protective immunity by inducing a memory response to an infectious
microorganism using an antigenic or immunogenic composition. Thus,
a vaccine is a composition capable of inducing a protective immune
response against a microorganism in a human being or animal. Such
an immune response can be a cellular response and/or a humoral
response, e.g. a specific T cell response or an antibody
response.
[0099] Accordingly, in an important embodiment, the composition is
a vaccine composition. I.e. the invention relates to the use of a
composition comprising [0100] a polypeptide which comprises a
sequence selected from the group consisting of surface-located
Streptococcus pneumoniae polypeptides of SEQ ID NO:1-282, or
comprises an antigenic fragment or variant of said sequence, [0101]
a polynucleotide comprising a sequence encoding said polypeptide,
[0102] an expression vector comprising a sequence encoding said
polypeptide, or [0103] a recombinant virus or recombinant cell
comprising said polynucleotide or said expression vector, as a
vaccine.
[0104] The variant herein preferably has at least 95% sequence
identity, such as at least 96%, e.g. at least 97%, such as at least
98%, e.g. at least 99% sequence identity to said sequence.
[0105] In one preferred embodiment of the above composition, the
polypeptide comprises SEQ ID NO:1, or an antigenic fragment or
variant thereof.
[0106] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:2, or an antigenic fragment or
variant thereof.
[0107] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:3, or an antigenic fragment or
variant thereof.
[0108] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:4, or an antigenic fragment or
variant thereof.
[0109] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:5, or an antigenic fragment or
variant thereof.
[0110] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:6, or an antigenic fragment or
variant thereof.
[0111] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:7, or an antigenic fragment or
variant thereof.
[0112] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:8, or an antigenic fragment or
variant thereof.
[0113] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:9, or an antigenic fragment or
variant thereof.
[0114] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:10, or an antigenic fragment or
variant thereof.
[0115] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:11, or an antigenic fragment or
variant thereof.
[0116] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:12, or an antigenic fragment or
variant thereof.
[0117] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:13, or an antigenic fragment or
variant thereof.
[0118] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:14, or an antigenic fragment or
variant thereof.
[0119] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:15, or an antigenic fragment or
variant thereof.
[0120] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:16, or an antigenic fragment or
variant thereof.
[0121] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:17, or an antigenic fragment or
variant thereof.
[0122] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:18, or an antigenic fragment or
variant thereof.
[0123] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:19, or an antigenic fragment or
variant thereof.
[0124] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:20, or an antigenic fragment or
variant thereof.
[0125] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:21, or an antigenic fragment or
variant thereof.
[0126] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:22, or an antigenic fragment or
variant thereof.
[0127] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:23, or an antigenic fragment or
variant thereof.
[0128] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:24, or an antigenic fragment or
variant thereof.
[0129] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:25, or an antigenic fragment or
variant thereof.
[0130] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:26, or an antigenic fragment or
variant thereof.
[0131] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:27, or an antigenic fragment or
variant thereof.
[0132] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:28, or an antigenic fragment or
variant thereof.
[0133] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:29, or an antigenic fragment or
variant thereof.
[0134] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:30, or an antigenic fragment or
variant thereof.
[0135] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:31, or an antigenic fragment or
variant thereof.
[0136] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:32, or an antigenic fragment or
variant thereof.
[0137] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:33, or an antigenic fragment or
variant thereof.
[0138] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:34, or an antigenic fragment or
variant thereof.
[0139] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:35, or an antigenic fragment or
variant thereof.
[0140] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:36, or an antigenic fragment or
variant thereof.
[0141] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:37, or an antigenic fragment or
variant thereof.
[0142] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:38, or an antigenic fragment or
variant thereof.
[0143] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:39, or an antigenic fragment or
variant thereof.
[0144] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:40, or an antigenic fragment or
variant thereof.
[0145] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:41, or an antigenic fragment or
variant thereof.
[0146] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:42, or an antigenic fragment or
variant thereof.
[0147] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:43, or an antigenic fragment or
variant thereof.
[0148] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:44, or an antigenic fragment or
variant thereof.
[0149] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:45, or an antigenic fragment or
variant thereof.
[0150] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:46, or an antigenic fragment or
variant thereof.
[0151] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:47, or an antigenic fragment or
variant thereof.
[0152] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:48, or an antigenic fragment or
variant thereof.
[0153] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:49, or an antigenic fragment or
variant thereof.
[0154] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:50, or an antigenic fragment or
variant thereof.
[0155] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:51, or an antigenic fragment or
variant thereof.
[0156] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:52, or an antigenic fragment or
variant thereof.
[0157] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:53, or an antigenic fragment or
variant thereof.
[0158] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:54, or an antigenic fragment or
variant thereof.
[0159] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:55, or an antigenic fragment or
variant thereof.
[0160] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:56, or an antigenic fragment or
variant thereof.
[0161] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:57, or an antigenic fragment or
variant thereof.
[0162] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:58, or an antigenic fragment or
variant thereof.
[0163] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:59, or an antigenic fragment or
variant thereof.
[0164] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:60, or an antigenic fragment or
variant thereof.
[0165] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:61, or an antigenic fragment or
variant thereof.
[0166] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:62, or an antigenic fragment or
variant thereof.
[0167] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:63, or an antigenic fragment or
variant thereof.
[0168] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:64, or an antigenic fragment or
variant thereof.
[0169] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:65, or an antigenic fragment or
variant thereof.
[0170] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:66, or an antigenic fragment or
variant thereof.
[0171] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:67, or an antigenic fragment or
variant thereof.
[0172] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:68, or an antigenic fragment or
variant thereof.
[0173] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:69, or an antigenic fragment or
variant thereof.
[0174] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:70, or an antigenic fragment or
variant thereof.
[0175] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:71, or an antigenic fragment or
variant thereof.
[0176] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:72, or an antigenic fragment or
variant thereof.
[0177] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:73, or an antigenic fragment or
variant thereof.
[0178] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:74, or an antigenic fragment or
variant thereof.
[0179] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:75, or an antigenic fragment or
variant thereof.
[0180] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:76, or an antigenic fragment or
variant thereof.
[0181] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:77, or an antigenic fragment or
variant thereof.
[0182] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:78, or an antigenic fragment or
variant thereof.
[0183] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:79, or an antigenic fragment or
variant thereof.
[0184] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:80, or an antigenic fragment or
variant thereof.
[0185] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:81, or an antigenic fragment or
variant thereof.
[0186] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:82, or an antigenic fragment or
variant thereof.
[0187] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:83, or an antigenic fragment or
variant thereof.
[0188] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:84, or an antigenic fragment or
variant thereof.
[0189] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:85, or an antigenic fragment or
variant thereof.
[0190] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:86, or an antigenic fragment or
variant thereof.
[0191] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:87, or an antigenic fragment or
variant thereof.
[0192] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:88, or an antigenic fragment or
variant thereof.
[0193] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:89, or an antigenic fragment or
variant thereof.
[0194] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:90, or an antigenic fragment or
variant thereof.
[0195] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:91, or an antigenic fragment or
variant thereof.
[0196] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:92, or an antigenic fragment or
variant thereof.
[0197] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:93, or an antigenic fragment or
variant thereof.
[0198] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:94, or an antigenic fragment or
variant thereof.
[0199] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:95, or an antigenic fragment or
variant thereof.
[0200] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:96, or an antigenic fragment or
variant thereof.
[0201] In another, preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:97, or an antigenic fragment or
variant thereof.
[0202] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:98, or an antigenic fragment or
variant thereof.
[0203] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:99, or an antigenic fragment or
variant thereof.
[0204] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:100, or an antigenic fragment
or variant thereof.
[0205] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:101, or an antigenic fragment
or variant thereof.
[0206] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:102, or an antigenic fragment
or variant thereof.
[0207] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:103, or an antigenic fragment
or variant thereof.
[0208] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:104, or an antigenic fragment
or variant thereof.
[0209] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:105, or an antigenic fragment
or variant thereof.
[0210] In another preferred embodiment of the above composition,
the polypeptide comprises SEQ ID NO:168, or an antigenic fragment
or variant thereof.
[0211] A composition comprising the polypeptide of SEQ ID NO:16, or
an antigenic fragment or variant thereof for use as a medicament is
at present the most preferred embodiment.
[0212] In some embodiments of the composition, the polypeptide
consists of a sequence selected from the group of SEQ ID NO:1-282.
In other embodiments, the polypeptide comprises a sequence selected
from the group of SEQ ID NO:1-282 or an antigenic fragment or
variant of said sequence, as well as a tag, such as a his-tag, i.e.
a polyhistidine tag.
[0213] In another preferred embodiment, the polypeptide in the
composition of the invention may be combined with or fused to a
toxin, e.g. an enterotoxigenic Escherichia coli Stable or Labile
toxin. A suitable heat stable toxin II (STII) has been described in
Lee et al. (1983) Infect. Immun. 42: 264-268. Examples of suitable
fusion proteins are given in SEQ ID NO:295 and SEQ ID NO:296. In
one embodiment, the combination comprises the polypeptide of the
invention and a non-covalently linked toxin, wherein the toxin may
be a single toxin polypeptide, or a multimeric, e.g. dimeric, form
comprising multiple copies of the toxin. In another embodiment, the
polypeptide of the invention and the toxin are covalently linked,
e.g. by post-translational linkage or transcription and translation
from a single fused open reading frame. In either case, the two
constituents may be linked directly or via a spacer or linker
domain, which e.g. may be a peptide linker, preferably a
protease-resistant and/or non-immunogenic peptide linker. Such
peptide linker may be of any length, e.g. it may be between 2 and
200, such as between 5 and 50 amino acids in length. Multiple
copies of the toxin may be fused to the polypeptide of the
invention.
[0214] A composition comprising a polypeptide of the invention,
e.g. the polypeptide of SEQ ID NO:16, as well as an enterotoxigenic
Escherichia coli may be used to manufacture a vaccine for
prevention of infection with Streptococcus pneumoniae and/or
enterotoxigenic Escherichia.
[0215] In further embodiments, the composition of the invention may
comprise dimers of any of the polypeptides of SEQ ID NO:1-282, such
as dimers of SEQ ID NO:16. Dimers may e.g. be formed by
post-translational linkage or be generated from a single fused open
reading frame. In either case, the two monomer units of the dimer
may be linked directly or via a spacer or linker domain, which e.g.
may be a peptide linker, preferably a protease-resistant and/or
non-immunogenic peptide linker. Such a peptide linker may be of any
length, e.g. it may be between 2 and 200, such as between 5 and 50
amino acids in length.
[0216] The composition may only comprise one polypeptide selected
from the group of SEQ ID NO:1-282 or a antigenic fragment or
variant thereof. However, in other embodiments, the composition
comprises more than one polypeptide of the group of SEQ ID NO:1-282
and/or more than one antigenic fragment of a polypeptide selected
from the group of SEQ ID NO:1-282. Thus, the composition according
to the invention may comprise more than one, such as 2, for example
3, such as 4, for example 5, such as 6, for example 7, such as 8,
for example 9, such as 10, such as a number of polypeptides and/or
fragments in the range of from 5 to 10, or more than 10, such as
for example in the range of from 10 to 20, different polypeptides
selected from the group of SEQ ID NO:1-282 or antigenic fragments
or variants thereof.
[0217] Similarly, the composition may only comprise one
polynucleotide, one expression vector or one recombinant virus or
recombinant cell of the invention. However, in other embodiments,
the composition comprises more than one polynucleotide, one
expression vector or one recombinant virus or recombinant cell of
the invention. Thus, the composition according to the invention may
comprise more than one, such as 2, for example 3, such as 4, for
example 5, such as 6, for example 7, such as 8, for example 9, such
as 10, or more than 10, such as for example in the range of from 10
to 20, different polynucleotides, expression vectors or recombinant
viruses or recombinant cells of the invention as described
herein.
[0218] Furthermore, in some embodiments, a recombinant cell of the
invention may express more than one polypeptide of the group of SEQ
ID NO:1-282 and/or more than one antigenic fragment or variant of a
polypeptide selected from the group of SEQ ID NO:1-282. Thus, the
composition according to the invention may comprise a recombinant
cell comprising more than one, such as 2, for example 3, such as 4,
for example 5, such as 6, for example 7, such as 8, for example 9,
such as 10, such as a number of polypeptides and/or antigenic
fragments or variants in the range of from 5 to 10, or more than
10, such as for example in the range of from 10 to 20, different
polypeptides selected from the group of SEQ ID NO:1-282 or
antigenic fragments or variants thereof. In another embodiment, the
composition for use in the invention comprises multiple of the
recombinant viruses or recombinant cells described herein.
[0219] Preferably, the composition of the invention comprises one
of the combinations of polypeptides (or antigenic fragments or
variants thereof) given in Table 1.
[0220] In Table 1, each of the crosses ("x") placed at the crossing
of a column designated with a SEQ ID number with a row designated
by another SEQ ID number indicates a composition comprising the two
polypeptides of those two SEQ ID numbers (or anti-genic fragments
or variants thereof).
[0221] I.e. as an example, entirely for illustrative purposes and
not intended in a limiting manner, the cross ("x") at the crossing
of the column of SEQ ID NO:2 ("2") with the row of SEQ ID NO:1
("1") indicates a composition comprising: [0222] the polypeptide of
SEQ ID NO: 1 or an antigenic fragment or variant thereof and [0223]
the polypeptide of SEQ ID NO:2 or an antigenic fragment or variant
thereof.
[0224] Highly preferred compositions include:
[0225] A composition comprising: [0226] the polypeptide of SEQ ID
NO:16 or an antigenic fragment or variant thereof and [0227] any of
the polypeptides of SEQ ID NO:1-282 or an antigenic fragment or
variant thereof, preferably any of SEQ ID NO:1-41 or an antigenic
fragment or variant thereof, more preferably a polypeptide selected
from the group consisting of SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:20 and SEQ ID NO:28, most preferably the polypeptide of SEQ ID
NO:20 or an antigenic fragment or variant thereof.
[0228] A composition comprising: [0229] the polypeptide of SEQ ID
NO:10 or an antigenic fragment or variant thereof and [0230] any of
the polypeptides of SEQ ID NO:1-282 or an antigenic fragment or
variant thereof, preferably any of SEQ ID NO:1-41 or an antigenic
fragment or variant thereof, more preferably a polypeptide selected
from the group consisting of SEQ ID NO:13, SEQ ID NO:20 and SEQ DI
NO:28, most preferably the polypeptide of SEQ ID NO:20 or an
antigenic fragment or variant thereof.
[0231] A composition comprising: [0232] the polypeptide of SEQ ID
NO:13 or an antigenic fragment or variant thereof and [0233] any of
the polypeptides of SEQ ID NO:1-282 or an antigenic fragment or
variant thereof, preferably any of SEQ ID NO:1-41 or an antigenic
fragment or variant thereof, more preferably a polypeptide selected
from the group consisting of SEQ ID NO:20 and SEQ ID NO:28, most
preferably the polypeptide of SEQ ID NO:20 or an antigenic fragment
or variant thereof.
[0234] A composition comprising: [0235] the polypeptide of SEQ ID
NO:28 or an antigenic fragment or variant thereof and [0236] any of
the polypeptides of SEQ ID NO:1-282 or an antigenic fragment or
variant thereof, preferably any of SEQ ID NO:1-41 or an antigenic
fragment or variant thereof, the polypeptide of SEQ ID NO:20 or an
antigenic fragment or variant thereof.
[0237] Preferred compositions comprising at least three
polypeptides include the following:
[0238] A composition comprising three or more polypeptides selected
from the group consisting of SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:16, SEQ ID NO:20 and SEQ ID NO:28.
[0239] A composition comprising: [0240] the polypeptide of SEQ ID
NO:16 or an antigenic fragment or variant thereof and [0241] the
polypeptide of SEQ ID NO:20 or an antigenic fragment or variant
thereof and [0242] any of the polypeptides of SEQ ID NO:1-282 or an
antigenic fragment or variant thereof, preferably any of SEQ ID
NO:1-41 or an antigenic fragment or variant thereof, more
preferably a polypeptide selected from the group consisting of SEQ
ID NO:10, SEQ ID NO:13 and SEQ ID NO:28.
[0243] Further preferred compositions according to the invention,
comprising four or more polypeptides selected from the group
consisting of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID
NO:20 and SEQ ID NO. 28.
[0244] In a yet further preferred embodiment, the composition of
the invention comprises the five polypeptides of SEQ ID NO:10, SEQ
ID NO:13, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO. 28.
[0245] In some embodiments of the above compositions comprising two
or more polypeptides, the polypeptides are not covalently linked.
In other embodiments, however, the polypeptides may form a fusion
polypeptide, which is formed by post-translational linkage or
generated from a single fused open reading frame. In either case,
the two or more polypeptides may be linked directly or via a spacer
or linker domain, which e.g. may be a peptide linker, preferably a
protease-resistant and/or non-immunogenic peptide linker. Such a
peptide linker may be of any length, e.g. it may be between 2 and
200, such as between 5 and 50 amino acids in length.
Vaccines Comprising Polypeptides
[0246] As described above, in a preferred embodiment, the invention
relates to a composition comprising a polypeptide which comprises a
sequence selected from the group consisting of SEQ ID NO:1-282, or
an antigenic fragment or variant of said sequence, for use as a
vaccine. Preferred fragments and variants are those described in
the sections herein that relate to fragments and variants.
[0247] Accordingly, in this embodiment, the antigenicity or
immunogenicity is provided by direct administration of a
polypeptide normally located on the surface of a Streptococcus
pneumoniae cell. In one particular embodiment, the polypeptides are
selected so that the vaccine composition comprises multiple
polypeptides capable of associating with different MHC molecules,
such as different MHC class I molecules. Preferably, the
composition for use as a vaccine comprises polypeptides and/or
fragments capable of associating with the most frequently occurring
MHC class I molecules. In one particular embodiment of the
invention, the composition comprises one or more polypeptides
and/or fragments capable of associating to an MHC class I molecule
and one or more polypeptides and/or fragments capable of
associating with an MHC class II molecule. Hence, the vaccine
composition is in some embodiments capable of raising a specific
cytotoxic T-cells response and/or a specific helper T-cell
response. Association to MHC molecules can e.g. be determined as
described by Andersen et al. (1999) Tissue Antigens 54:185; or by
Tan et al. (1997) J. Immunol. Methods 209:25.
Adjuvants and Immunogenic Carriers
[0248] Preferably, the composition for use as vaccine, i.e. the
vaccine composition, of the present invention comprises a
pharmaceutically-acceptable carrier as described herein in the
section `Compositions for use in the invention`.
[0249] The composition can further comprise an adjuvant. Adjuvants
are substances whose admixture into the vaccine composition
increases or otherwise modifies the immune response to a
polypeptide or other antigen. Adjuvants could for example be any
of: AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2, AlNH.sub.4
(SO.sub.4), silica, alum, Al(OH).sub.3, Ca.sub.3 (PO.sub.4).sub.2,
kaolin, carbon, aluminium hydroxide, aluminium phosphate, muramyl
dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),
N-acetyl-nornuramyl-Lalanyl-D-iso-glutamine (CGP 11687, also
referred to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-
-glycero-3-hydroxphosphoryl oxy)-ethylamine (CGP 19835A, also
referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2%
squalene/Tween-80.RTM. emulsion, lipopolysaccharides and
derivatives, including lipid A, Freund's Complete Adjuvant (FCA),
Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides
(for example, poly IC and poly AU acids), wax D from Mycobacterium,
tuberculosis, substances found in Corynebacterium parvum,
Bordetella pertussis, and members of the genus Brucella, liposomes
or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S.
Pat. Nos. 58,767 and 5,554,372), Lipid A derivatives, choleratoxin
derivatives, HSP derivatives, LPS derivatives, synthetic peptide
matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51
and QS-21. Preferred adjuvants to be used with the invention
include alum, Montanide ISA-51 and QS-21. Montanide ISA-51 (Seppic,
Inc.) is a mineral oil-based adjuvant analogous to incomplete
Freund's adjuvant, which is normally administered as an emulsion.
QS-21 (Antigenics; Aquila Biopharmaceuticals, Framingham, Mass.) is
a highly purified, water-soluble saponin that handles as an aqueous
solution. Another preferred adjuvant to be used in the composition
of the invention is IMSAVAC-L from the Netherlands Vaccine
Institute. In another preferred embodiment, the polypeptide or
polypeptides are included in virosomes.
[0250] Desirable functionalities of adjuvants capable of being used
in accordance with the present invention are listed in the below
table.
TABLE-US-00003 TABLE 1 Modes of adjuvant action Action Adjuvant
type Benefit 1. Immunomodulation Generally small molecules or
proteins which modify the Upregulation of immune response.
Selection of Th1 or cytokine network Th2 2. Presentation Generally
amphipathic molecules or complexes which Increased neutralizing
antibody response. Greater interact with immunogen in its native
conformation duration of response 3. CTL Induction Particles which
can bind or enclose immunogen and Cytosolic processing of protein
yielding correct class which can fuse with or disrupt cell
membranes 1 restricted peptides w/o emulsions for direct attachment
of peptide to cell Simple process if promiscuous peptide(s) known
surface MHC-1 4. Targeting Particulate adjuvants which bind
immunogen. Adjuvants Efficient use of adjuvant and immunogen which
saturate Kupffer cells Carbohydrate adjuvants which target lectin
receptors on As above. May also determine type of response if
macrophages and DCs targeting selective 5. Depot generation w/o
emulsion for short term Efficiency Microspheres or nanospheres for
long term Potential for single-dose vaccine
[0251] Source: John C. Cox and Alan R. Coulter Vaccine 1997
February; 15(3):248-56 A vaccine composition according to the
present invention may comprise more than one different adjuvant. It
is also contemplated that the Streptococcus pneumoniae polypeptide
of the invention, or one or more antigenic fragments thereof, and
the adjuvant can be administered separately in any appropriate
sequence.
[0252] The adjuvant of choice may e.g. Freund's complete or
incomplete adjuvant, or killed B. pertussis organisms, used e.g. in
combination with alum precipitated antigen. A general discussion of
adjuvants is provided in Goding, Monoclonal Antibodies: Principles
& Practice (2nd edition, 1986) at pages 61-63. Goding notes,
however, that when the antigen of interest is of low molecular
weight, or is poorly immunogenic, coupling to an immunogenic
carrier is recommended (see below). Various saponin extracts and
cytokines have also been suggested to be useful as adjuvants in
immunogenic compositions. Recently, it has been proposed to use
granulocyte-macrophage colony stimulating factor (GM-CSF), a well
known cytokine, as an adjuvant (WO 97/28816).
[0253] In addition, a vaccine composition of the invention can
comprise an immunogenic carrier such as a scaffold structure, for
example a protein or a polysaccharide, to which the Streptococcus
pneumoniae polypeptide or the fragment thereof is capable of being
associated. A Streptococcus pneumoniae polypeptide, or the
antigenic fragment or variant thereof, present in the vaccine
composition can thus be associated with an immunogenic carrier such
as e.g. a protein. The binding or association of the polypeptide to
a carrier protein may be covalent or non-covalent. An immunogenic
carrier protein may be present independently of an adjuvant. The
function of a carrier protein can for example be to increase the
molecular weight of in particular fragments in order to increase
their activity or immunogenicity, to confer stability, to increase
the biological activity, or to increase serum half-life.
Furthermore, an immunogenic carrier protein may aid presenting the
Streptococcus pneumoniae polypeptide or the fragments thereof to T
cells. A carrier protein could be, but is not limited to, keyhole
limpet hemocyanin, serum proteins such as transferrin, bovine serum
albumin, human serum albumin, thyroglobulin or ovalbumin,
Immunoglobulins, or hormones, such as insulin. Tetanus toxoid
and/or diphtheria toxoid are also suitable carriers in one
embodiment of the invention. Alternatively or additionally,
dextrans, for example sepharose may be added. In yet another
embodiment, an antigen-presenting cell such as e.g. a dendritic
cell capable of presenting the polypeptide or a fragment thereof to
a T cell may be added to obtain the same effect as a carrier
protein. Methods for the preparation of vaccine compositions have
e.g. been described in U.S. Pat. No. 5,470,958 and references
therein.
[0254] In a further embodiment, the vaccine composition of the
invention may comprise Streptococcus pneumoniae carbohydrates in
addition to a polypeptide of the invention. In one embodiment, the
added carbohydrates are carbohydrates derived from or
characteristic of one or more serotypes of Streptococcus
pneumoniae. In a preferred embodiment, the polypeptide of the
invention is combined with polysaccharides derived from or
characteristic of any one or more of the serotypes given in Table
4. In a preferred embodiment, the polypeptide is combined with one
or more, preferably two, three, four, five, six or seven
polysaccharides derived from or characteristic of serotype 4, 6B,
9V, 14, 18C, 19F and 23F. In another embodiment, the polypeptide is
combined with eight or more, preferably ten or more, 15 or more, or
20 or more of the polysaccharide antigens of serotypes 1, 2, 3, 4,
5, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,
22F, 23F, and 33F. These carbohydrates may added in free form to
the vaccine composition of the invention, or, alternatively, they
may be fused to a polypeptide of the invention to be used in the
vaccine composition.
[0255] An effective amount of a polypeptide of the invention may be
an amount capable of eliciting a detectable humoral immune response
in the absence of an immunomodulator. The appropriate amount of
immunogen to be used is dependent on the immunological response it
is desired to elicit. Furthermore, the exact effective amount
necessary may vary from subject to subject, depending on the
species, age and general condition of the subject, the severity of
the condition being treated, the mode of administration, etc. The
polypeptide vaccines of the present invention may be administered
in various dosages, including dosages that are lower than those
normally used for other vaccines. This possible because the
polypeptides of the present invention are abundant on the surface
of a Streptococcus pneumoniae cell and thus even a fairly low level
of response can provide immunity. Thus, dosage of a polypeptide of
the invention, when used for immunisation, may e.g. be from 0.1 to
500 micrograms per kilogram body weight, such as from 0.1 to 100
micrograms, e.g. from 0.1 to 50 micrograms, such as from 0.1 to 25
micrograms, such as in the range of from 8 to 25 micrograms per
kilogram body weight, or less than that, such as from 0.1 to 5
micrograms or from 0.1 to 2 micrograms per kilograms body
weight.
DNA Vaccine Compositions and Vaccine Compositions Comprising
Recombinant Viruses or Recombinant Cells
[0256] DNA or RNA vaccines pertain to the introduction of e.g. an
antigenic polypeptide determinant into a patient by overexpressing
in the cells of the patient, a polynucleotide construct which
includes expression control sequences operably linked to a sequence
encoding the polypeptide of interest, herein a polypeptide of any
of SEQ ID NO:1-282 or an antigenic fragment or variant thereof,
preferably the polypeptide of SEQ ID NO:16 or an antigenic fragment
or variant thereof. As such fragments may not contain a methionine
start codon, such a codon is optionally included as part of the
expression control sequences. The polynucleotide construct may be a
non-replicating and linear polynucleotide, a circular expression
vector, or an autonomously replicating plasmid or viral expression
vector. The construct may become integrated into the host genome.
Any expression vector that can transfect a mammalian cell may be
used in the methods of immunising an individual according to the
present invention. Methods for constructing expression vectors are
well known in the art (see, e.g., Molecular Cloning: A Laboratory
Manual, Sambrook et al., eds., Cold Spring Harbor Laboratory, 2nd
Edition, Cold Spring Harbor, N.Y., 1989). Preferred are
compositions comprising a plurality of genes expressing multiple
polypeptides selected from SEQ ID NO:1-282 and/or multiple
antigenic fragments of the invention, thereby permitting
simultaneous vaccination using a variety of preselected
targets.
[0257] Vaccines can also be prepared by incorporating a
polynucleotide encoding a specific antigenic polypeptide of
interest into a living but harmless vector, such as a virus or a
cell, such as an attenuated or reduced-virulence E. coli or
Salmonella cell. The harmless recombinant virus or recombinant cell
is injected into the intended recipient. Such a recombinant cell
may be dead or alive. If alive, the recombinant organism may
replicate in the host while producing and presenting the antigenic
polypeptide to the host's immune system. It is contemplated that
this type of vaccine may be more effective than the non-replicative
type of vaccine. For such a vaccine to be successful, the vector
organism must be viable, and either be naturally non-virulent or
have an attenuated or reduced-virulence phenotype.
[0258] Strategies for vaccination using attenuated bacteria and
suitable bacterial strains for use therein have been described in
e.g. Makino et al. (2001) Microb. Pathog. 31:1-8; Gentschev et al.
(2002) Int. J. Med. Microbiol. 291:577-582; Turner et al. (2001)
Infect. Immun. 69:4969-4979; WO99/49026; and WO03/022307.
[0259] Further examples of vectors that can be applied are vectors
comprising e.g., retroviruses, as disclosed in WO 90/07936, WO
91/02805, WO 93/25234, WO 93/25698, and WO 94/03622, adenovirus, as
disclosed by Berkner, Biotechniques 6:616-627, 1988; Li et al.,
Hum. Gene Ther. 4:403-409, 1993; Vincent et al., Nat. Genet.
5:130-134, 1993; and Kolls et al., Proc. Natl. Acad. Sci. USA
91:215-219, 1994), pox virus, as disclosed by U.S. Pat. No.
4,769,330; U.S. Pat. No. 5,017,487; and WO 89/01973, naked DNA as
disclosed WO 90/11092, a polynucleotide molecule complexed to a
polycationic molecule as disclosed in WO 93/03709, and
polynucleotides associated with liposomes as disclosed by Wang et
al., Proc. Natl. Acad. Sci. USA 84:7851, 1987. In certain
embodiments, the DNA may be linked to killed or inactivated
adenovirus as disclosed by Curiel et al., Hum. Gene Ther.
3:147-154, 1992; Cotton et al., Proc. Natl. Acad. Sci. USA 89:6094,
1992. Other suitable compositions include DNA-ligands as disclosed
by Wu et al., J. Biol. Chem. 264:16985-16987, 1989), and lipid-DNA
combinations as disclosed by Felgner et al., Proc. Natl. Acad. Sci.
USA 84:7413-7417, 1989). In addition, the efficiency of naked DNA
uptake into cells may be increased by coating the DNA onto
biodegradable latex beads.
[0260] Vaccine vectors preferably comprise a suitable promoter
which is operably linked to the polynucleotide sequence encoding
the immunogenic polypeptide. Any promoter that can direct a high
level of transcription initiation in the target cells may be used
in the invention. Non-tissue specific promoters, such as the
cytomegalovirus (DeBemardi et al., Proc Natl Acad Sci USA
88:9257-9261 [1991], and references therein), mouse metallothionine
I (Hammer et al., J Mol Appl Gen 1:273-288 [1982]), HSV thymidine
kinase (McKnight, Cell 31:355-365 [1982]), and SV40 early (Benoist
et al., Nature 290:304-310 [1981]) promoters may thus also be
used.
Methods of Vaccination and Use for Vaccination/Immunisation
[0261] In a further main aspect, the present invention relates to
the use of a composition comprising any one or more of [0262] a
polypeptide which comprises a sequence selected from the group
consisting of SEQ ID NO:1-282, or comprises an antigenic fragment
or variant of said sequence, [0263] a polynucleotide comprising a
sequence encoding said polypeptide, [0264] an expression vector
comprising a sequence encoding said polypeptide, or [0265] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, for the preparation of a
medicament for the immunisation of an animal or human being against
bacterial infections. The immunisation preferably induces a
protective immune response. In one embodiment of the above use, the
medicament is only given once.
[0266] In a preferred embodiment, the medicament is for the
immunisation against Streptococcus infections. Most preferably, the
medicament is for immunisation against Streptococcus pneumoniae.
Immunisation with a Streptococcus pneumoniae polypeptide can,
however, also give cross-protection to other bacterial species.
This normally requires significant homology to at least a portion
of a polypeptide of the other species. Such homology is e.g. found
between SEQ ID NO:16 and variants thereof from Streptococcus
pyogenes (group A Streptococcus) (SEQ ID NO:283), Streptococcus
agalactiae (group B Streptococcus) (SEQ ID NO:284) and Listeria
monocytogenes (SEQ ID NO:285). Similarly, homology is found between
SEQ ID NO:20 and variants thereof from Streptococcus pyogenes
(group A Streptococcus) (SEQ ID NO:286), Streptococcus agalactiae
(group B Streptococcus) (SEQ ID NO:287) and Listeria monocytogenes
(SEQ ID NO:288).
[0267] Accordingly, the medicament is in some embodiments used for
the immunisation against one or more of: Streptococcus pyogenes
(group A Streptococcus), Streptococcus agalactiae (group B
Streptococcus) and Listeria monocytogenes. Highly preferred
polypeptides for use in the preparation of such a medicament are
SEQ ID NO:16 and SEQ ID NO:20.
[0268] An alternative strategy for immunisation against one or more
bacteria is to immunise with a medicament comprising the variant
polypeptide. Accordingly, in a further embodiment, the polypeptide
used for the preparation of the medicament is a variant of any of
SEQ ID NO:1-282, preferably a variant of SEQ ID NO:16 and/or a
variant of SEQ ID NO:20. Most preferably, the polypeptide is
selected from the group consisting of SEQ ID NO:283, SEQ ID NO:284,
SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287 and SEQ ID NO:288, or a
fragment thereof or a variant thereof, e.g. a variant having more
than 95%, such as more than 98% sequence identity to SEQ ID NO:283,
SEQ ID NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287 or SEQ
ID NO:288.
[0269] Accordingly, in some embodiments: [0270] a medicament
comprising SEQ ID NO:283 and/or SEQ ID NO:286, or a fragment or
variant of any of these two, is used to immunise against
Streptococcus pyogenes and/or Streptococcus pneumoniae and/or other
bacteria; [0271] a medicament comprising SEQ ID NO:284 and/or SEQ
ID NO:287, or a fragment or a variant of any of these two, is used
to immunise against Streptococcus agalactiae and/or Streptococcus
pneumoniae and/or other bacteria or [0272] a medicament comprising
SEQ ID NO:285 and/or SEQ ID NO:288, or a fragment or a variant of
any of these two, is used immunise against Listeria monocytogenes
and/or Streptococcus pneumoniae and/or other bacteria
[0273] In the most preferred embodiment, the composition herein
comprises or further comprises [0274] a polypeptide which comprises
SEQ ID NO:16, or comprises an antigenic fragment or variant of SEQ
ID NO:16, [0275] a polynucleotide comprising a sequence encoding
said polypeptide, [0276] an expression vector comprising a sequence
encoding said polypeptide, or [0277] a recombinant virus or
recombinant cell comprising said polynucleotide or said expression
vector.
[0278] In another preferred embodiment, the composition herein
comprises or further comprises [0279] a polypeptide which comprises
SEQ ID NO:10, or comprises an antigenic fragment or variant of SEQ
ID NO:10, [0280] a polynucleotide comprising a sequence encoding
said polypeptide, [0281] an expression vector comprising a sequence
encoding said polypeptide, or a recombinant virus or recombinant
cell comprising said polynucleotide or said expression vector.
[0282] In another preferred embodiment, the composition herein
comprises or further comprises [0283] a polypeptide which comprises
SEQ ID NO:13, or comprises an antigenic fragment or variant of SEQ
ID NO:13, [0284] a polynucleotide comprising a sequence encoding
said polypeptide, [0285] an expression vector comprising a sequence
encoding said polypeptide, or [0286] a recombinant virus or
recombinant cell comprising said polynucleotide or said expression
vector.
[0287] In another preferred embodiment, the composition herein
comprises or further comprises [0288] a polypeptide which comprises
SEQ ID NO:28, or comprises an antigenic fragment or variant of SEQ
ID NO:28, [0289] a polynucleotide comprising a sequence encoding
said polypeptide, [0290] an expression vector comprising a sequence
encoding said polypeptide, or [0291] a recombinant virus or
recombinant cell comprising said polynucleotide or said expression
vector.
[0292] In another preferred embodiment, the composition further
comprises [0293] a polypeptide which comprises SEQ ID NO:20, or
comprises an antigenic fragment or variant of SEQ ID NO:20, [0294]
a polynucleotide comprising a sequence encoding said polypeptide,
[0295] an expression vector comprising a sequence encoding said
polypeptide, or [0296] a recombinant virus or recombinant cell
comprising said polynucleotide or said expression vector.
[0297] Similarly, the invention relates to a method for the
immunisation of an animal or human being against a Streptococcus
pneumoniae infections comprising the step of administering any one
or more of [0298] a polypeptide which comprises any of the
sequences of SEQ ID NO:1-282, or comprises a fragment or variant of
any of said sequences, [0299] a polynucleotide comprising a
sequence encoding said polypeptide, [0300] an expression vector
comprising a sequence encoding said polypeptide, or [0301] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, thereby immunising said
animal or human being against Streptococcus pneumoniae
infections.
[0302] In one embodiment of the above method for immunisation, said
[0303] polypeptide which comprises any of the sequences of SEQ ID
NO:1-282, preferably SEQ ID NO:16, or comprises a fragment or
variant of any of said sequences, [0304] polynucleotide comprising
a sequence encoding said polypeptide, [0305] expression vector
comprising a sequence encoding said polypeptide, or [0306]
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, is only given once,
thereby immunising said animal or human being against Streptococcus
pneumoniae infections through a single administration. The animal
may be any bird or mammal, e.g. a chicken, duck, turkey, cow or
pig. Particular target populations of human beings may be
individuals from at-risk populations, such as the population of
children up to 4 years old, the population of elderly persons or
the population of naive or semi-immune travelers to the developing
world.
[0307] Because the polypeptides of the present invention are
immunogenic and because they are abundant on the Streptococcus
pneumoniae cell, a protective immune response can be induced even
patients with a reduced ability to respond to antigenic stimuli,
such as juveniles, elderly patients or immunocompromised patients.
Furthermore, for the same reasons, the vaccines of the invention
can also be used to prevent otitis media, to prevent nasopharyngal
carriage of Streptococcus pneumoniae, to prevent sepsis caused by
Streptococcus pneumoniae, or to prevent meningitis caused by
Streptococcus pneumoniae.
[0308] Thus, in one embodiment, the present invention relates to
the use of any one or more of [0309] a polypeptide which comprises
a sequence selected from the group consisting of SEQ ID NO:1-282,
preferably SEQ ID NO:16, or comprises an antigenic fragment or
variant of said sequence, [0310] a polynucleotide comprising a
sequence encoding said polypeptide, [0311] an expression vector
comprising a sequence encoding said polypeptide, or [0312] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, for the preparation of a
medicament for the immunisation of an animal or human being against
Streptococcus pneumoniae infections, wherein said human being is a
child of less than 4 yours of age, such as less than 2 years of
age, e.g. less than 1 year of age, and/or a child having maternal
immunity (i.e. having maternal antibodies in circulation).
[0313] In a further embodiment, the present invention relates to
the use of any one or more of [0314] a polypeptide which comprises
a sequence selected from the group consisting of SEQ ID NO:1-282,
preferably SEQ ID NO:16, or comprises an antigenic fragment or
variant of said sequence, [0315] a polynucleotide comprising a
sequence encoding said polypeptide, [0316] an expression vector
comprising a sequence encoding said polypeptide, or [0317] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, for the preparation of a
medicament for the immunisation of an animal or human being against
Streptococcus pneumoniae infections, wherein said human being is an
immunocompromised patient. Immunocompromised patients could e.g.
patients taking immunosuppressive chemotherapy or patients with
congenital or acquired immune deficiency. For the immunisation to
be effective in these patients, it is required that the patient
still to some extent is capable of producing an immune
response.
[0318] In another embodiment, the present invention relates to the
use of any one or more of [0319] a polypeptide which comprises a
sequence selected from the group consisting of SEQ ID NO:1-282,
preferably SEQ ID NO:16, or comprises an antigenic fragment or
variant of said sequence, [0320] a polynucleotide comprising a
sequence encoding said polypeptide, [0321] an expression vector
comprising a sequence encoding said polypeptide, or a recombinant
virus or recombinant cell comprising said polynucleotide or said
expression vector, for the preparation of a medicament for the
prevention of otitis media, in particular otitis media due to
Streptococcus pneumoniae.
[0322] In yet another embodiment, the present invention relates to
the use of any one or more of [0323] a polypeptide which comprises
a sequence selected from the group consisting of SEQ ID NO:1-282,
preferably SEQ ID NO:16, or comprises an antigenic fragment or
variant of said sequence, [0324] a polynucleotide comprising a
sequence encoding said polypeptide, [0325] an expression vector
comprising a sequence encoding said polypeptide, or [0326] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, for the preparation of a
medicament for the treatment and/or prevention of nasopharyngal
carriage of Streptococcus pneumoniae.
[0327] In an even further embodiment, the present invention relates
to the use of any one or more of [0328] a polypeptide which
comprises a sequence selected from the group consisting of SEQ ID
NO:1-282, preferably SEQ ID NO:16, or comprises an antigenic
fragment or variant of said sequence, [0329] a polynucleotide
comprising a sequence encoding said polypeptide, [0330] an
expression vector comprising a sequence encoding said polypeptide,
or [0331] a recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, for the preparation of a
medicament for the prevention of Streptococcal meningitis.
[0332] The vaccines may be administered in the dosages described
herein by any suitable mode of administration, including modes of
administration that result in a less than complete (e.g. less than
50% or less than 90%) uptake of all administered antigen. This is
possible because the polypeptides of the present invention are
sufficiently immunogenic and because, due to their abundance on the
surface of the Streptococcus pneumoniae cell, even a somewhat
suboptimal response, can provide immunity. Thus, modes of
administration of the composition according to the invention
include, but are not limited to systemic administration, such as
intravenous or subcutaneous administration, transdermal
administration, intradermal administration, intramuscular
administration, intranasal administration, oral administration, and
generally any form of mucosal administration.
[0333] An important problem relating to the production of effective
Streptococcus vaccines is the occurrence of immunologically
different types, also termed serotypes, of the bacteria. These
types differ considerably in their polysaccharide profile and also,
albeit less, in some highly variable proteins. Due to such
variability, vaccines known in the art often only work against some
and not all serotypes.
[0334] The vaccines of the present invention are based on abundant
surface-located polypeptides, which are not highly variable. These
vaccines will be effective against a plurality of serotypes.
Accordingly, in one embodiment, the invention relates to the use of
any one or more of [0335] a polypeptide which comprises a sequence
selected from the group consisting of SEQ ID NO:1-282, preferably
selected from the group consisting of SEQ ID NO:1-41, most
preferably SEQ ID NO:16, or comprises an antigenic fragment or
variant of said sequence, [0336] a polynucleotide comprising a
sequence encoding said polypeptide, [0337] an expression vector
comprising a sequence encoding said polypeptide, or [0338] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector, for the preparation of a
medicament for the immunisation of an animal or human being against
more than one serotype of Streptococcus pneumoniae, such as 5 or
more different serotypes, e.g. 8 or more different serotypes, such
as 15 or more different serotypes, e.g. 24 or more different
serotypes.
[0339] Preferably, said more than one serotype includes a serotype
selected from the group of 6A, 7C, 9A, 10B, 13, 15C, 16F, 18B, 21,
23A, 24F, 28F, 31, 34, 35F, 35B, 38.
[0340] In one preferred embodiment, the medicament is used for the
immunisation against at least the serotypes 4, 6B, 9V, 14, 18C, 19F
and 23F, and, preferably, furthermore at least one further
serotype, said further serotype preferably being selected from the
group of 6A, 7C, 9A, 10B, 13, 15C, 16F, 18B, 21, 23A, 24F, 28F, 31,
34, 35F, 35B, 38.
[0341] In another preferred embodiment, the medicament is used for
the immunization against at least the serotypes 1, 2, 3, 4, 5, 6B,
7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F,
23F, and 33F, and preferably at least one further serotype,
preferably selected from the group of 6A, 7C, 9A, 10B, 13, 15C,
16F, 18B, 21, 23A, 24F, 28F, 31, 34, 35F, 35B, 38.
[0342] In a further preferred embodiment, the medicament is used
for the immunisation against any of the serotypes given in Table 4,
preferably at least 5 or more different serotypes selected from the
serotypes given in Table 4, e.g. 8 or more different serotypes,
such as 15 or more different serotypes, e.g. 24 or more different
serotypes selected from the serotypes given in Table 4.
[0343] The immunogenic effect according to the present invention
can e.g. be measured by assay of antibodies in serum samples e.g.
by a RIA. Furthermore, the effect can be determined in vivo, by
measuring e.g. an increased T-cell responsiveness to T-cell
dependent antigenic polypeptides, wherein said increased
responsiveness is characteristic of an enhancement of a normal
immune response to such antigenic polypeptides. An
immunostimulating effect may also be measured as an enhanced T cell
production of, in particular, IL-2, IL-3, IFN-.gamma. and/or
GM-CSF. Polypeptides or fragments thereof having a potential for
eliciting an enhanced immune response may thus be readily
identified by screening for enhanced IL-2, IL-3, IFN-.gamma. or
GM-CSF production by T cells, as described e.g. in U.S. Ser. No.
07/779,499, incorporated herein by reference.
[0344] A number of aspects related to vaccination against
Streptococcus pneumoniae have been discussed in Bogaert et al.
(2004) Vaccine 22:2209-2220. This review includes references to
other documents describing methods for testing and evaluation of
such vaccines.
[0345] The herein described polynucleotides and expression vectors
can be introduced into target cells in vivo or in vitro by any
standard method: e.g., as naked DNA (Donnelly et al., Annu Rev
Immunol 15:617-648 [1997]), incorporated into ISCOMS, liposomes, or
erythrocyte ghosts, or by biolistic transfer, calcium
precipitation, or electroporation. Alternatively, one can employ a
viral-based vector as a means for introducing the polynucleotide
encoding the polypeptide of interest Into the cells of the animal
or human being. Preferred viral vectors include those derived from
replication-defective hepatitis viruses (e.g., HBV and HCV),
retroviruses (see, e.g., WO89/07136; and Rosenberg et al., N Eng J
Med 323 (9):570-578 [1990]), adenovirus (see, e.g., Morsey et al.,
J Cell Biochem, Supp. 17E [1993]), adeno-associated virus (Kotin et
al., Proc Natl Acad Sci USA 87:2211-2215 [1990]), replication
defective herpes simplex viruses (HSV; Lu et al., Abstract, page
66, Abstracts of the Meeting on Gene Therapy, Sep. 22-26, 1992,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), canary
pox virus, and any modified versions of these vectors. Cells
transfected in vitro can be cultured and cloned, if desired, prior
to introduction into the patient.
[0346] In addition to direct in vivo procedures, ex vivo procedures
may be used in which cells are removed from an animal, modified,
and placed into the same or another animal. It will be evident that
one can utilise any of the compositions noted above for
introduction of an antigenic polypeptides or polynucleotides
encoding such according to the invention into tissue cells in an ex
vivo context. Protocols for viral, physical and chemical methods of
uptake are well known in the art. Thus, as an alternative to
administration of a polypeptide of the invention or a vector
capable of expressing such a polypeptide directly to the patient,
one can remove helper T cells from the patient; stimulate those T
cells ex vivo using the same antigenic polypeptide or vector; and
introduce the stimulated helper T cells into the same patient.
Antibodies and Methods for Raising Antibodies of the Invention
[0347] In a further main embodiment, the composition for use as a
medicament comprises an antibody capable of binding a polypeptide
selected from the group consisting of surface-located Streptococcus
pneumoniae polypeptides of SEQ ID NO:1-282, preferably selected
from the group consisting of SEQ ID NO:1-41, more preferably
selected from the group consisting of SEQ ID NO:16, SEQ ID NO:10,
SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID
NO:33, most preferably the polypeptide of SEQ ID NO:16. Such a
medicament can be used for antibody therapy, such as passive
immunisation of an individual in need thereof.
[0348] Accordingly, in a further main aspect, the invention relates
to antibodies capable of binding, preferably specifically binding,
a polypeptide selected from the group consisting of SEQ ID NO:1-282
and/or a fragment and/or a variant thereof `Specifically binding`
is, in this context, not intended to mean absolute specificity. The
antibody may in some embodiments also specifically bind
polypeptides, e.g. from other Streptococcus species, with a high
degree of sequence identity to the polypeptide from Streptococcus
pneumoniae, e.g. polypeptides with more than 90%, such as more than
95% or more than 98% sequence identity to the polypeptide from
Streptococcus pneumoniae.
[0349] In a preferred embodiment, the antibody is capable of
binding, preferably specifically binding, a polypeptide selected
from the group consisting of SEQ ID NO:1-282, such as the
polypeptide of SEQ ID NO:1, for example the polypeptide of SEQ ID
NO:2, such as the polypeptide of SEQ ID NO:3, for example the
polypeptide of SEQ ID NO:4, such as the polypeptide of SEQ ID NO:5,
for example the polypeptide of SEQ ID NO:6, such as the polypeptide
of SEQ ID NO:7, for example the polypeptide of SEQ ID NO:8, such as
the polypeptide of SEQ ID NO:9, for example the polypeptide of SEQ
ID NO:10, such as the polypeptide of SEQ ID NO:11, for example the
polypeptide of SEQ ID NO:12, such as the polypeptide of SEQ ID
NO:13, for example the polypeptide of SEQ ID NO:14, such as the
polypeptide of SEQ ID NO:15, for example the polypeptide of SEQ ID
NO:16, such as the polypeptide of SEQ ID NO:17, for example the
polypeptide of SEQ ID NO:18, such as the polypeptide of SEQ ID
NO:19, for example the polypeptide of SEQ ID NO:20, such as the
polypeptide of SEQ ID NO:21, for example the polypeptide of SEQ ID
NO:22, such as the polypeptide of SEQ ID NO:23, for example the
polypeptide of SEQ ID NO:24, such as the polypeptide of SEQ ID
NO:25, for example the polypeptide of SEQ ID NO:26, such as the
polypeptide of SEQ ID NO:27, for example the polypeptide of SEQ ID
NO:28, such as the polypeptide of SEQ ID NO:29, for example the
polypeptide of SEQ ID NO:30, such as the polypeptide of SEQ ID
NO:31, for example the polypeptide of SEQ ID NO:32, such as the
polypeptide of SEQ ID NO:33, for example the polypeptide of SEQ ID
NO:34, such as the polypeptide of SEQ ID NO:35, for example the
polypeptide of SEQ ID NO:36, such as the polypeptide of SEQ ID
NO:37, for example the polypeptide of SEQ ID NO:38, such as the
polypeptide of SEQ ID NO:39, for example the polypeptide of SEQ ID
NO:40, such as the polypeptide of SEQ ID NO:41, for example the
polypeptide of SEQ ID NO:42, such as the polypeptide of SEQ ID
NO:43, for example the polypeptide of SEQ ID NO:44, such as the
polypeptide of SEQ ID NO:45, for example the polypeptide of SEQ ID
NO:46, such as the polypeptide of SEQ ID NO:47, for example the
polypeptide of SEQ ID NO:48, such as the polypeptide of SEQ ID
NO:49, for example the polypeptide of SEQ ID NO:50, such as the
polypeptide of SEQ ID NO:51, for example the polypeptide of SEQ ID
NO:52, such as the polypeptide of SEQ ID NO:53, for example the
polypeptide of SEQ ID NO:54, such as the polypeptide of SEQ ID
NO:55, for example the polypeptide of SEQ ID NO:56, such as the
polypeptide of SEQ ID NO:57, for example the polypeptide of SEQ ID
NO:58, such as the polypeptide of SEQ ID NO:59, for example the
polypeptide of SEQ ID NO:60, such as the polypeptide of SEQ ID
NO:61, for example the polypeptide of SEQ ID NO:62, such as the
polypeptide of SEQ ID NO:63, for example the polypeptide of SEQ ID
NO:64, such as the polypeptide of SEQ ID NO:65, for example the
polypeptide of SEQ ID NO:66, such as the polypeptide of SEQ ID
NO:67, for example the polypeptide of SEQ ID NO:68, such as the
polypeptide of SEQ ID. NO:69, for example the polypeptide of SEQ ID
NO:70, such as the polypeptide of SEQ ID NO:71, for example the
polypeptide of SEQ ID NO:72, such as the polypeptide of SEQ ID
NO:73, for example the polypeptide of SEQ ID NO:74, such as the
polypeptide of SEQ ID NO:75, for example the polypeptide of SEQ ID
NO:76, such as the polypeptide of SEQ ID NO:77, for example the
polypeptide of SEQ ID NO:78, such as the polypeptide of SEQ ID
NO:79, for example the polypeptide of SEQ ID NO:80, such as the
polypeptide of SEQ ID NO:81, for example the polypeptide of SEQ ID
NO:82, such as the polypeptide of SEQ ID NO:83, for example the
polypeptide of SEQ ID NO:84, such as the polypeptide of SEQ ID
NO:85, for example the polypeptide of SEQ ID NO:86, such as the
polypeptide of SEQ ID NO:87, for example the polypeptide of SEQ ID
NO:88, such as the polypeptide of SEQ ID NO:89, for example the
polypeptide of SEQ ID NO:90, such as the polypeptide of SEQ ID
NO:91, for example the polypeptide of SEQ ID NO:92, such as the
polypeptide of SEQ ID NO:93, for example the polypeptide of SEQ ID
NO:94, such as the polypeptide of SEQ ID NO:95, for example the
polypeptide of SEQ ID NO:96, such as the polypeptide of SEQ ID
NO:97, for example the polypeptide of SEQ ID NO:98, such as the
polypeptide of SEQ ID NO:99, for example the polypeptide of SEQ ID
NO:100, such as the polypeptide of SEQ ID NO:101, for example the
polypeptide of SEQ ID NO:102, such as the polypeptide of SEQ ID
NO:103, for example the polypeptide of SEQ ID NO:104, such as the
polypeptide of SEQ ID NO:105, for example the polypeptide of SEQ ID
NO:168.
[0350] In preferred embodiments, the antibodies of the invention
are furthermore capable of binding an intact Streptococcus
pneumoniae cell, i.e. capable of binding a living or a dead
Streptococcus cell which has maintained its structural integrity,
preferably a cell that has maintained the integrity of the membrane
(i.e. wherein the membrane has not been permeabilized). Binding of
antibodies to intact cells can e.g. be determined by flow cytometry
as described in Rioux et al. (2001) Infect. Immun. 69:5162-5165 or
as described in Singh et al. (2003) Infect. Immun.
71:3937-3946.
[0351] Preferred antibodies are ones that bind with a dissociation
constant or Kd of less than 5.times.10.sup.-8M, such as less than
10.sup.-6M, e.g. less than 5.times.10.sup.-7M, such as less than
10.sup.-7M, e.g. less than 5.times.10.sup.-8M, such as less than
10.sup.-8M, e.g. less than 5.times.10.sup.-9M, such as less than
10.sup.-9M, e.g. less than 5.times.10.sup.-10M, such as less than
10.sup.-10M, e.g. less than 5.times.10.sup.-11M, such as less than
10.sup.-11M, e.g. less than 5.times.10.sup.-12M, such as less than
10.sup.-12M, e.g. less than 5.times.10.sup.-13M, such as less than
10.sup.-13M, e.g. less than 5.times.10.sup.-14M, such as less than
10.sup.-14M, e.g. less than 5.times.10.sup.-15M, or less than
10.sup.-15M. Binding constants can be determined using methods
well-known in the art, such as ELISA (e.g. as described in Orosz
and Ovadi (2002) J. Immunol. Methods 270:155-162) or surface
plasmon resonance analysis.
[0352] Antibodies can be used for passive immunisation of mammals,
preferably human beings, more preferably immunocompromised
patients. A treatment with antibodies can be done to cure or to
prevent Streptococcus pneumoniae infections, including pneumococcal
diseases, such as pneumonia or meningitis or pneumococcal sepsis.
Preferred patient groups include children under the age of 4 years,
elderly patients or immunocompromised patients.
[0353] Antibodies of the invention include the following preferred
mechanistic groups: [0354] 1. Function-inhibiting antibodies that
work as an antibacterial (affect the viability of the bacterium).
Such antibodies should be effective regardless of the immune status
of the patient. Preferably, such antibodies are capable of reducing
Streptococcus pneumoniae growth in vitro to less than 50%, such as
less than 25%, for example less than 10%, such as less than 5% of a
control without antibody added. [0355] 2. Opsonising antibodies
that are designed to enhance phagocytic killing. Effectiveness of
such antibodies may depend on the immune status of the patient, but
it is very well possible that they will enhance phagocytic killing
even in compromised patients. [0356] 3. Antibodies conjugated to a
therapeutic moiety such as a toxin or bactericidal agent, e.g.
ricin or radioisotopes. Techniques for conjugating a therapeutic
moiety to antibodies are well known, see, e.g. Thorpe et al. (1982)
Immunol. Rev. 62, 119-158. These antibodies should also be
effective regardless of the immune status of the patient. An
antibody with or without a therapeutic moiety conjugated to it can
be used as a therapeutic that is administered alone or in
combination with chemotherapeutics or other therapeutic agents.
[0357] In one embodiment, the antibodies of the invention are
opsonising as well as function-inhibiting. In another embodiment,
the antibodies of the invention are opsonising, but not
function-inhibiting. The latter group of antibodies can e.g. be
antibodies directed against a target polypeptide which is not
essential for the viability of Streptococcus pneumoniae.
[0358] In a further main aspect, the invention relates to a method
for raising antibodies to a polypeptide selected from the group
consisting of SEQ ID NO:1-282, in a non-human animal comprising the
steps of [0359] a. providing [0360] a polypeptide comprising a
sequence selected from the group consisting of SEQ ID NO:1-282,
preferably selected from the group consisting of SEQ ID NO:16, SEQ
ID NO:10, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28
and SEQ ID NO:33, most preferably SEQ ID NO:16, or comprising an
antigenic fragment or variant of said sequence, [0361] a
polynucleotide comprising a sequence encoding said polypeptide,
[0362] an expression vector comprising a sequence encoding said
polypeptide, [0363] or [0364] a recombinant virus or recombinant
cell comprising said polynucleotide or said expression vector,
[0365] b. introducing a composition comprising said polypeptide,
polynucleotide, vector, recombinant virus or recombinant cell into
said animal, [0366] c. raising antibodies in said animal, [0367] d.
isolating and optionally purifying the antibodies.
[0368] In a preferred embodiment, antibodies capable of binding an
intact Streptococcus pneumoniae cell are identified by comprising
performing the above steps and the further step of selecting
antibodies capable of binding an intact Streptococcus pneumoniae
cell.
[0369] The above methods are preferably done in a transgenic animal
which is capable of producing human antibodies. In a further
preferred embodiment, the above methods are non-therapeutic.
Monoclonal/Polyclonal Antibodies
[0370] Antibodies of the invention may be polyclonal antibodies or
monoclonal antibodies or mixtures of monoclonal antibodies. In a
preferred embodiment, the antibody is a monoclonal antibody or a
fragment thereof. Monoclonal antibodies (Mab's) are anti-bodies
wherein every antibody molecule is similar and thus recognises the
same epitope. The antibody may be any kind of antibody, however, it
is preferably an IgG or IgA antibody.
[0371] Preferred antibodies, more preferably monoclonal antibodies,
are antibodies capable of specifically binding surface-exposed
regions of the polypeptides of the invention. Accordingly, in a
preferred embodiment of an antibody capable of binding SEQ ID
NO:16, said antibody binds an epitope on SEQ ID NO:16 which
comprises one or more amino acids of any of SEQ ID NO:289-SEQ ID
NO:294. Even more preferably, said antibody binds an epitope which
comprises two or more, such as three or more, e.g., four or more,
such as five or more amino acids of a sequence selected from the
group consisting of SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291,
SEQ ID NO:292, SEQ ID NO:293 and SEQ ID NO:294.
[0372] Monoclonal antibodies are in general produced by a hybridoma
cell line. Methods of making monoclonal antibodies and
antibody-synthesising hybridoma cells are well known to those
skilled in the art. Antibody-producing hybridomas may for example
be prepared by fusion of an antibody-producing B lymphocyte with an
immortalised cell line. A monoclonal antibody can be produced by
the following steps. An animal is immunised with an antigen such as
a full-length polypeptide or a fragment thereof. The immunisation
is typically accomplished by administering the antigen to an
immunologically competent mammal in an immunologically effective
amount, i.e., an amount sufficient to produce an immune response.
Preferably, the mammal is a rodent such as a rabbit, rat or mouse.
The mammal is then maintained on a booster schedule for a time
period sufficient for the mammal to generate high affinity antibody
molecules. A suspension of antibody-producing cells is removed from
each immunised mammal secreting the desired antibody. After a
sufficient time to generate high affinity antibodies, the animal
(e.g. mouse) is sacrificed and antibody-producing lymphocytes are
obtained from one or more of the lymph nodes, spleens and
peripheral blood. Spleen cells are preferred, and can be
mechanically separated into individual cells in a physiological
medium using methods well known to one of skill in the art. The
antibody-producing cells are immortalised by fusion to cells of a
mouse myeloma line. Mouse lymphocytes give a high percentage of
stable fusions with mouse homologous myelomas, however, rat, rabbit
and frog somatic cells can also be used. Spleen cells of the
desired antibody-producing animals are immortalised by fusing with
myeloma cells, generally in the presence of a fusing agent such as
polyethylene glycol. Any of a number of myeloma cell lines suitable
as a fusion partner can be, for example, the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O--Ag14 myeloma lines, available from the
American Type Culture Collection (ATCC), Rockville, Md.
[0373] Monoclonal antibodies can also be generated by other methods
well known to those skilled in the art of recombinant DNA
technology. An alternative method, referred to as the
"combinatorial antibody display" method, has been developed to
identify and isolate antibody fragments having a particular
specificity, and can be utilised to produce monoclonal
antibodies.
[0374] A polyclonal antibody is a mixture of antibody molecules
recognising a specific given antigen, hence polyclonal antibodies
may recognise different epitopes within e.g. a polypeptide. In
general polyclonal antibodies are purified from serum of a mammal,
which previously has been immunised with the antigen. Polyclonal
antibodies may for example be prepared by any of the methods
described in Antibodies: A Laboratory Manual, By Ed Harlow and
David Lane, Cold Spring Harbor Laboratory Press, 1988. Polyclonal
antibodies may be derived from any suitable mammalian species, for
example from mice, rats, rabbits, donkeys, goats, and sheep.
Specificity
[0375] The antibodies of the invention may be monospecific towards
any of the polypeptides of SEQ ID NO:1-282. In another embodiment,
the antibody is bispecific or multispecific having at least one
portion being specific towards any of the polypeptides of SEQ ID
NO:1-282.
[0376] Monospecific antibodies may be monovalent, i.e. having only
one binding domain. For a monovalent antibody, the immunoglobulin
constant domain amino-acid sequences preferably comprise the
structural portions of an antibody molecule known in the art as
CH1, CH2, CH3 and CH4. Preferred are those which are known in the
art as C.sub.L. Furthermore, insofar as the constant domain can be
either a heavy or light chain constant domain (C.sub.H or C.sub.L,
respectively), a variety of monovalent antibody compositions are
contemplated by the present invention. For example, light chain
constant domains are capable of disulphide bridging to either
another light chain constant domain, or to a heavy chain constant
domain. In contrast, a heavy chain constant domain can form two
independent disulphide bridges, allowing for the possibility of
bridging to both another heavy chain and to a light chain, or to
form polymers of heavy chains. Thus, in another embodiment, the
invention contemplates a composition comprising a monovalent
polypeptide wherein the constant chain domain C has a cysteine
residue capable of forming at least one disulphide bridge, and
where the composition comprises at least two monovalent
polypeptides covalently linked by said disulphide bridge.
[0377] In another embodiment of the invention the antibody is a
multivalent antibody having at least two binding domains. The
binding domains may have specificity for the same ligand or for
different ligands.
Multispecificity, Including Bispecificity
[0378] In a preferred embodiment the invention relates to
multispecific antibodies, which have affinity for and are capable
of specifically binding at least two different entities.
[0379] In one embodiment, the multispecific antibody is a
bispecific antibody, which carries at least two different binding
domains, at least one of which is of antibody origin. A bispecific
molecule of the invention can also be a single chain bispecific
molecule. Multispecific molecules can also be single-chain
molecules or may comprise at least two single-chain molecules. The
multispecific, including bispecific antibodies, may be produced by
any suitable manner known to the person skilled in the art. A
number of approaches have been developed such as the ones described
in WO 94/09131; WO 94/13804; WO 94/13806 or U.S. Pat. Nos.
5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;
5,013,653; 5,258,498; and 5,482,858. Using a bispecific or
multispecific antibody according to the invention the invention
offers several advantages as compared to monospecific/monovalent
antibodies. A bispecific/multispecific antibody has a first binding
domain capable of specifically recognising and binding any of the
Streptococcus pneumoniae polypeptides of SEQ ID NO:1-282, whereas
the other binding domain(s) may be used for other purposes. In one
embodiment, at least one other binding domain is used for binding
to a Streptococcus pneumoniae polypeptide, such as binding to
another epitope on the same Streptococcus pneumoniae polypeptide as
the first binding domain. Thereby specificity for Streptococcus
pneumoniae may be increased as well as increase of avidity of the
antibody. In another embodiment the at least one other binding
domain may be used for specifically binding a mammalian cell, such
as a human cell. It is preferred that the at least other binding
domain is capable of binding an immunoactive cell, such as a
leukocyte, a macrophage, a lymphocyte, a basophilic cell, and/or an
eosinophilic cell, in order to increase the effect of the antibody
in a therapeutic method. This may be accomplished by establishing
that the at least one other binding domain is capable of
specifically binding a mammalian protein, such as a human protein,
such as a protein selected from any of the cluster differentiation
proteins (CD), in particular CD64 and/or CD89.
Humanised Antibodies
[0380] It is not always desirable to use non-human antibodies for
human therapy, since the non-human "foreign" epitopes may elicit an
immune response in the individual to be treated. To eliminate or
minimise the problems associated with non-human antibodies, it is
desirable to engineer chimeric antibody derivatives, i.e.,
"humanised" antibody molecules that combine the non-human Fab
variable region binding determinants with a human constant region
(Fc). Such antibodies are characterised by equivalent antigen
specificity and affinity of the monoclonal and polyclonal
antibodies described above, and are less immunogenic when
administered to humans, and therefore more likely to be tolerated
by the individual to be treated.
[0381] Accordingly, in one embodiment the antibody of the invention
is a humanised antibody. Humanised antibodies are in general
chimeric antibodies comprising regions derived from a human
antibody and regions derived from a non-human antibody, such as a
rodent antibody. Humanisation (also called Reshaping or
CDR-grafting) is a well-established technique for reducing the
immunogenicity of monoclonal antibodies (mAbs) from xenogeneic
sources (commonly rodent), increasing the homology to a human
immunoglobulin, and for improving their activation of the human
immune system. Thus, humanised antibodies are typically human
antibodies in which some CDR residues and possibly some framework
residues are substituted by residues from analogous sites in rodent
antibodies.
[0382] It is important that humanised antibodies retain high
affinity for the antigen and other favourable biological
properties. To achieve this goal, according to a preferred method,
humanised antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanised products using
three-dimensional models of the parental and humanised sequences.
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of certain residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is maximised, although it is the CDR residues that
directly and most substantially influence antigen binding.
[0383] One method for humanising MAbs relates to production of
chimeric antibodies in which an antigen binding site comprising the
complete variable domains of one antibody is fused to constant
domains derived from a second antibody, preferably a human
antibody. Methods for carrying out such chimerisation procedures
are for example described in EP-A-0 120 694 (Celltech Limited),
EP-A-0 125 023 (Genentech Inc.), EP-A-0 171 496 (Res. Dev. Corp.
Japan), EP-A-0173494 (Stanford University) and EP-A-0 194 276
(Celltech Limited).
[0384] The humanised antibody of the present invention may be made
by any method capable of replacing at least a portion of a CDR of a
human antibody with a CDR derived from a non-human antibody. Winter
describes a method which may be used to prepare the humanised
antibodies of the present invention (UK Patent Application GB
2188638A), the contents of which are incorporated by reference.
[0385] As an example, the humanised antibodies of the present
invention may be produced by the following process: [0386] (a)
constructing, by conventional techniques, an expression vector
containing an operon with a DNA sequence encoding an antibody heavy
chain in which the CDRs and such minimal portions of the variable
domain framework region that are required to retain antibody
binding specificity are derived from a non-human immunoglobulin,
and the remaining parts of the antibody chain are derived from a
human immunoglobulin; [0387] (b) constructing, by conventional
techniques, an expression vector containing an operon with a DNA
sequence encoding a complementary antibody light chain in which the
CDRs and such minimal portions of the variable domain framework
region that are required to retain donor antibody binding
specificity are derived from a non-human immunoglobulin, and the
remaining parts of the antibody chain are derived from a human
immunoglobulin; [0388] (c) transfecting the expression vectors into
a host cell by conventional techniques; and [0389] (d) culturing
the transfected cell by conventional techniques to produce the
humanised antibody.
[0390] The host cell may be co-transfected with the two vectors of
the invention, the first vector containing an operon encoding a
light chain derived polypeptide and the second vector containing an
operon encoding a heavy chain derived polypeptide. The two vectors
contain different selectable markers, but otherwise, apart from the
antibody heavy and light chain coding sequences, are preferably
identical, to ensure, as far as possible, equal expression of the
heavy and light chain polypeptides. Alternatively, a single vector
may be used, the vector including the sequences encoding both the
light and the heavy chain polypeptides. The coding sequences for
the light and heavy chains may comprise cDNA or genomic DNA or
both.
[0391] The host cell used to express the altered antibody of the
invention may be either a bacterial cell such as Escherichia coli,
or a eukaryotic cell. In particular a mammalian cell of a well
defined type for this purpose, such as a myeloma cell or a Chinese
hamster ovary cell may be used.
[0392] The general methods by which the vectors of the invention
may be constructed, transfection methods required to produce the
host cell of the invention and culture methods required to produce
the antibody of the invention from such host cells are all
conventional techniques. Likewise, once produced, the humanised
antibodies of the invention may be purified according to standard
procedures.
Human Antibodies
[0393] In a more preferred embodiment the invention relates to an
antibody, Wherein the binding domain is carried by a human
antibody, i.e. wherein the antibodies have a greater degree of
human peptide sequences than do humanised antibodies.
[0394] Human mAb antibodies directed against human proteins can be
generated using transgenic mice carrying the human immune system
rather than the mouse system. Splenocytes from these transgenic
mice immunised with the antigen of interest are used to produce
hybridomas that secrete human mAbs with specific affinities for
epitopes from a human protein (see, e.g., Wood et al. International
Application WO 91/00906, Kucherlapati et al. PCT publication WO
91/10741; Lonberg et al. International Application WO 92/03918; Kay
et al. International Application 92/03917; Lonberg, N. et al. 1994
Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21;
Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA
81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon
et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol
21:1323-1326). Such transgenic mice are available from Abgenix,
Inc., Fremont, Calif., and Medarex, Inc., Annandale, N.J. It has
been described that the homozygous deletion of the antibody
heavy-chain joining region (1H) gene in chimeric and germ-line
mutant mice results in complete inhibition of endogenous antibody
production. Transfer of the human germ-line immunoglobulin gene
array in such germ-line mutant mice will result in the production
of human anti-bodies upon antigen challenge. See, e.g., Jakobovits
et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et
al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.
7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human
antibodies can also be derived from phage-display libraries
(Hoogenboom et al., J. Mol. Biol. 227: 381 (1992); Marks et al., J.
Mol. Biol. 222:581-597 (1991); Vaughan, et al., Nature Biotech
14:309 (1996)).
[0395] A preferred method for the isolation of high affinity
antibodies is a subtractive procedure were human antibodies or
antibody fragments against the targets, in particular against the
antigens 029 (SEQ ID NO:16) and 607 (SEQ ID NO:20), in their native
configuration can be rapidly obtained from a phage antibody library
(see, e.g. De Kruif et al., Proc. Natl. Acad. Sci. USA 92:3938-3942
(1995); U.S. Pat. No. 6,265,150; and US patent applications
2002132228 and 2005043521). The phage antibody libraries can e.g.
be constructed using antibody producing cells from patients with
the disease of interest, here patients infected with Streptococcus
pneumoniae. The genes coding for the antibodies produced by these
cells may be cloned into a semi-synthetic phage antibody library
using degenerated oligonucleotides rearranging the CDR3 Region of
the cloned genes. Afterwards the library is incubated with the
target antigen or target-expressing cells, here Streptococcus
pneumoniae, and the phage antibodies bound to the target are
isolated by using standard methods. The present invention is also
directed to antibodies identified by the above procedure, in
particular antibodies capable of binding a polypeptide selected
from group consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13,
SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:33,
preferably antibodies having a dissociation constant or Kd of less
than 10.sup.-7M, e.g. less than 10.sup.-8M, such as less than
10.sup.-9M, e.g. less than 10.sup.-10M, such as less than
10.sup.-11M and/or antibodies binding a surface-exposed epitope of
any of these targets.
[0396] Suitable methods for producing human monoclonal antibodies
have furthermore been described in WO 03/017935, WO 02/100348, US
2003 091561, and US 2003 194403.
Binding Fragments of Antibodies
[0397] In one embodiment of the invention, the antibody is a
fragment of an antibody, preferably an antigen binding fragment or
a variable region. Examples of antibody fragments useful with the
present invention include Fab, Fab', F(ab').sub.2 and Fv fragments.
Papain digestion of antibodies produces two identical antigen
binding fragments, called the Fab fragment, each with a single
antigen binding site, and a residual "Fc" fragment, so-called for
its ability to crystallise readily. Pepsin treatment yields an
F(ab').sub.2 fragment that has two antigen binding fragments which
are capable of cross-linking antigen, and a residual other fragment
(which is termed pFc'). Additional fragments can include diabodies,
linear antibodies, single-chain antibody molecules, and
multispecific antibodies formed from antibody fragments.
[0398] The antibody fragments Fab, Fv and scFv differ from whole
antibodies in that the antibody fragments carry only a single
antigen-binding site. Recombinant fragments with two binding sites
have been made in several ways, for example, by chemical
cross-linking of cysteine residues introduced at the C-terminus of
the VH of an Fv (Cumber et al., 1992), or at the C-terminus of the
VL of an scFv (Pack and Pluckthun, 1992), or through the hinge
cysteine residues of Fab's (Carter et al., 1992).
[0399] Preferred antibody fragments retain some or essentially all
of the ability of an antibody to selectively binding with its
antigen. Some preferred fragments are defined as follows: [0400]
(1) Fab is the fragment that contains a monovalent antigen-binding
fragment of an antibody molecule. A Fab fragment can be produced by
digestion of whole anti-body with the enzyme papain to yield an
intact light chain and a portion of one heavy chain. [0401] (2)
Fab' is the fragment of an antibody molecule and can be obtained by
treating whole antibody with pepsin, followed by reduction, to
yield an intact light chain and a portion of the heavy chain. Two
Fab' fragments are obtained per antibody molecule. Fab' fragments
differ from Fab fragments by the addition of a few residues at the
carboxyl terminus of the heavy chain CH1 domain including one or
more cysteines from the antibody hinge region. [0402] (3)
(Fab').sub.2 is the fragment of an antibody that can be obtained by
treating whole anti-body with the enzyme pepsin without subsequent
reduction. F(ab').sub.2 is a dimer of two Fab' fragments held
together by two disulfide bonds. [0403] (4) Fv is the minimum
antibody fragment that contains a complete antigen recognition and
binding site. This region consists of a dimer of one heavy and one
light chain variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). It is in this configuration that the three
CDRs of each variable domain interact to define an antigen binding
site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the
six CDRs confer antigen binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for an antigen) has the ability to
recognise and bind antigen, although at a lower affinity than the
entire binding site.
[0404] In one embodiment of the present invention the antibody is a
single-chain antibody, defined as a genetically engineered molecule
containing the variable region of the light chain, the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule. Such single-chain
antibodies are also referred to as "single-chain Fv" or "sFv"
antibody fragments. Generally, the Fv polypeptide further comprises
a polypeptide linker between the V.sub.H and V.sub.L domains that
enables the sFv to form the desired structure for antigen
binding.
[0405] The antibody fragments according to the invention may be
produced in any suitable manner known to the person skilled in the
art. Several microbial expression systems have already been
developed for producing active antibody fragments, e.g. the
production of Fab in various hosts, such as E. coli or yeast has
been described, The fragments can be produced as Fab's or as Fv's,
but additionally it has been shown that a V.sub.H and a V.sub.L can
be genetically linked in either order by a flexible polypeptide
linker, which combination is known as an scFv.
Compositions for Use in the Invention
[0406] In a preferred embodiment of the composition for use as a
medicament, said composition comprises, in addition to the active
component, a pharmaceutically-acceptable carrier.
[0407] As used herein, the term "pharmaceutically acceptable" used
in connection with compositions or carriers represents that the
materials are capable of being administered to or upon a human or
animal without the production of undesirable physiological effects
such as nausea, dizziness, gastric upset and the like.
[0408] The preparation of a composition that contains active
ingredients dissolved or dispersed therein is well understood in
the art. Often such compositions are prepared as sterile
injectables either as liquid solutions or suspensions, aqueous or
non-aqueous, however, solid forms suitable for solution, or
suspension, in liquid prior to use can also be prepared. The
preparation can also be emulsified. The active ingredient can be
mixed with carriers which are pharmaceutically acceptable and
compatible with the active ingredient and in amounts suitable for
use in the methods described herein. Suitable carriers are, for
example, water, saline, dextrose, glycerol, ethanol or the like and
combinations thereof. In addition, if desired, the composition can
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH-buffering agents and the like which enhance
the effectiveness of the active ingredient.
[0409] The compositions of the present invention can include
pharmaceutically-acceptable salts of the active components therein.
Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the polypeptide) that are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, tartaric,
mandelic and the like. Salts formed with the free carboxyl groups
can also be derived from inorganic bases such as, for example,
sodium, potassium, ammonium, calcium or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine, procaine and the like.
[0410] Pharmaceutically-acceptable carriers are well known in the
art. Exemplary of liquid carriers are sterile aqueous solutions
that contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH value, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, propylene glycol, polyethylene
glycol and other solutes. Liquid compositions can also contain
liquid phases in addition to and to the exclusion of water.
Exemplary of such additional liquid phases are glycerin, vegetable
oils such as cottonseed oil, organic esters such as ethyl oleate,
and water-oil emulsions.
[0411] The composition may also be a kit-in-part further including
an antibiotic agent, such as antibiotics selected from vancomycin,
.beta.-lactams, cephalosporins, penicillins, aminoglycosides,
macrolide antibiotics (erythromycin, clarithromycin, or
azithromycin) and fluoroquinolone antibiotics (ciprofloxacin,
levofloxacin, gatifloxacin, or moxifloxacin) and/or including an
immunostimulating agent, such as cytokines, interferons, growth
factors, for example GCSF or GM-CSF. The kit-in-part may be used
for simultaneous, sequential or separate administration.
[0412] The Invention furthermore relates to pharmaceutical
compositions useful for practising the methods described herein.
Thus, the invention relates to a pharmaceutical composition
comprising a pharmaceutically-acceptable carrier and [0413] an
isolated polypeptide which comprises any of the sequences of SEQ ID
NO:1-282, preferably selected from the group consisting of SEQ ID
NO:16, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:20, and SEQ ID NO:28,
most preferably SEQ ID NO:16, or comprises a fragment or variant of
any of said sequences, [0414] an isolated polynucleotide comprising
a sequence encoding said polypeptide, [0415] an expression vector
comprising a sequence encoding said polypeptide, [0416] or [0417] a
recombinant virus or recombinant cell comprising said
polynucleotide or said expression vector.
[0418] Furthermore, the invention relates to a pharmaceutical
composition comprising an antibody of the invention, preferably an
antibody capable of binding a polypeptide selected from the group
consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:33, most preferably
SEQ ID NO:16, as defined herein and a pharmaceutically-acceptable
carrier.
Polypeptides of the Invention
Fragments of the Invention
[0419] In a further aspect, the invention relates to a fragment,
preferably an antigenic fragment, of a polypeptide set forth in any
of SEQ ID NO:1-282, preferably SEQ ID NO:16. Antigenicity can be
predicted by various methods known in the art. The length of such
fragments may vary from 2 consecutive amino-acid residues of a
polypeptide to the full-length polypeptide minus one amino-acid
residue. Preferably, fragments are less than 100 consecutive amino
acids, such as less than 70 or 50 consecutive amino acids, e.g.
less than consecutive 40 amino acids, such as less than 30
consecutive amino acids, e.g. less than 25 consecutive amino acids,
such as less than consecutive 20 amino acids in length. Thus, for
example fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19 or 20 consecutive amino acids in length. In
further preferred embodiments, a fragment comprises 6 or more, such
as 7 or more, e.g. 8 or more, such as 9 or more, e.g. 10 or more
consecutive amino acids of the corresponding full-length sequence.
Preferred ranges include fragments of between 5 and 50 consecutive
amino acids in length, such as between 5 and 25 consecutive amino
acids in length, e.g. between 5 and 20 consecutive amino acids in
length. Expressed in another way, a fragment consists of a part of
an amino-acid sequence which is less than 100% in length as
compared to the full-length polypeptide. Preferably, the length of
the fragment is less than 99%, such as less than 75%, e.g. less
than 50%, such as less than 25%, e.g. less than 20%, such as less
than 15%, e.g. less than 10% of the length of the full-length
polypeptide. In further preferred embodiments, the fragment
consists of a part of an amino-acid sequence which is less than
100%, but more than 1% in length as compared to the full-length
polypeptide, such as less than 100% but more than 5%, e.g. less
than 100% but more than 10%, such as less than 100% but more than
20%, e.g. less than 100% but more than 25%, such as less than 100%
but more than 50% of the length of the full-length polypeptide.
[0420] Preferably, fragments of the invention are surface-exposed
in an intact Streptococcus pneumoniae cell or other cell when
expressed recombinantly therein. Surface-exposure can be e.g. be
determined using a monoclonal antibody specific for said fragment,
e.g. as described in Singh et al. (2003) Infect. Immun.
71:3973-3946. Also preferred are fragments which are capable of
inducing antibodies that can specifically bind an intact
Streptococcus pneumoniae cell. This can be determined by generating
monoclonal antibodies using said fragment and subsequent
characterisation of the binding of individual antibodies to Intact
cells, e.g. as described in Singh et al. (2003) Infect. Immun.
71:3973-3946. Preferred fragments of SEQ ID NO:16 include fragments
comprising or consisting of one or more of the sequences of SEQ ID
NO:289-SEQ ID NO:294, more preferred fragments include fragments
comprising or consisting of SEQ ID NO:289 and/or SEQ ID NO:290,
fragments comprising or consisting of SEQ ID NO: 291 and/or SEQ ID
NO: 292, and fragments comprising or consisting of SEQ ID NO:293
and/or SEQ ID NO:294.
[0421] The full-length polypeptides of SEQ ID NO:1-282 as well as
the fragments of the invention can be produced recombinantly by
conventional techniques known in the art. Suitable host cells can
be mammalian cells, e.g. CHO, COS or HEK293 cells. Alternatively,
insect cells, bacterial cells or fungal cells can be used. Methods
for heterologous expression of polynucleotide sequences in the cell
types listed above and subsequent purification of the produced
polypeptides, e.g. using a tag sequence such as a histidine tag,
which may be removed after purification, are well-known to those
skilled in the art. Alternatively, fragments of the invention can
be produced synthetically.
Variants of the Invention
[0422] In a further main aspect, the invention relates to the use
of variants of any of the polypeptides set forth in SEQ ID
NO:1-282, preferably SEQ ID NO:16, or variants of fragments of any
of the polypeptides set forth in SEQ ID NO:1-282, preferably SEQ ID
NO:16, in a composition for use as a medicament. When used herein,
phrases such as `a polypeptide having at least 95% sequence
identity to SEQ ID NO:X` are used interchangeably with, and are
intended to be directed to the same subject-matter as, phrases such
as `the polypeptide of SEQ ID NO:X and variants thereof, wherein
the variant has at least 95% sequence identity to said
sequence.`
[0423] Variants preferably have at least 75% sequence identity, for
example at least 80% sequence identity, such as at least 85%
sequence identity, for example at least 90% sequence identity, such
as at least 91% sequence identity, such as at least 92% sequence
identity, for example at least 93% sequence identity, such as at
least 94% sequence identity, for example at least 95% sequence
identity, such as at least 96% sequence identity, for example at
least 97% sequence identity, such as at least 98% sequence
Identity, for example 99% sequence identity with the given
polypeptide or fragment. Sequence identity is determined with any
of the algorithms GAP, BESTFIT, or FASTA in the Wisconsin Genetics
Software Package Release 7.0, using default gap weights.
[0424] Preferred variants of a given polypeptide or fragment are
variants in which all amino-acid substitutions between the variant
and the given reference polypeptide or fragment are conservative
substitutions. Conservative amino-acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine, a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino-acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0425] Variants of a polypeptide or of a fragment thereof also
include forms of the polypeptide or fragment wherein one or more
amino acids have been deleted or inserted. Preferably, less than 5,
such as less than 4, e.g. less than 3, such as less than 2, e.g.
only one amino acid has been inserted or deleted. `Variants` of a
polypeptide or of a fragment thereof also include forms of these
polypeptides or fragments modified by post-translational
modifications of the amino-acid sequence.
Recombinant Cells of the Invention
[0426] In a further main aspect, the invention relates to the use
of a recombinant cell transformed or transfected with a
polynucleotide comprising a sequence encoding a polypeptide, said
polypeptide comprising a sequence selected from the group
consisting of SEQ ID NO:1-282, preferably SEQ ID NO:16, or
comprising an antigenic fragment or variant of said sequence.
Preferably, said recombinant cell is an Escherichia coli or
Salmonella cell, more preferably an attenuated or reduced-virulence
Escherichia or Salmonella cell.
[0427] Suitable bacterial strains for use herein have been
described in e.g. Makino et al. (2001) Microb. Pathog. 31:1-8;
Gentschev et al. (2002) Int. J. Med. Microbiol. 291:577-582; Turner
et al. (2001) Infect. Immun. 69:4969-4979; WO99/49026; and
WO03/022307 and references therein. Examples of suitable Salmonella
strains are CvD908-T7pol (Santiago-Machuca et al. (2002) Plasmid
47:108-119), ATCC 39183, ATCC 53647 and ATCC 53648. Examples of
suitable E. coli strains are YT106 and E1392/75-2A.
METHODS AND USES OF THE INVENTION
[0428] The compositions and other products defined above can be
used to treat or prevent Streptococcus pneumoniae infections,
and/or disease resulting from such infections, In animals or human
beings in need thereof.
[0429] Treatment and prevention herein include all types of
therapeutic treatment and preventive treatment and other treatments
to combat Streptococcus pneumoniae, including but not limited to
vaccination, prophylaxis, active immunisation, passive
immunisation, administration of antibodies, curative treatment,
ameliorating treatment. In particular, passive immunisation using
antibodies of the invention is a suitable treatment for
immunocompromised individuals.
Diagnostic Methods of the Invention
[0430] The combination of being surface-exposed and being present
in relatively high copy numbers in cells also makes the
polypeptides identified by the inventors highly suitable as targets
for detection of Streptococcus pneumoniae, allowing detection of
this organism with high sensitivity.
[0431] Accordingly, in a further main aspect, the invention relates
to a method for detecting Streptococcus pneumoniae or parts thereof
in a sample comprising the steps of [0432] a. contacting said
sample with an indicator moiety capable of specifically binding a
polypeptide selected from the group consisting of SEQ ID NO:1-282,
preferably selected from the group consisting of SEQ ID NO:16, SEQ
ID NO:10, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28
and SEQ ID NO:33, most preferably SEQ ID NO:16, and [0433] b.
determining whether a signal has been generated by the indicator
moiety, thereby detecting whether said sample contains
Streptococcus pneumoniae or parts thereof. Preferably, said
indicator moiety is capable of binding, preferably specifically
binding, intact Streptococcus pneumoniae cells.
[0434] In preferred embodiments of the above diagnostic methods, a
washing step is performed between the contacting step and the
determination step, in order to improve the specificity of
detection.
[0435] The sample can e.g. be feces, urine, a tissue, tissue
extract, fluid sample or body fluid sample, such as blood, plasma,
serum, sputum, or a sample taken from nose or lung. Another example
of a sample is a food sample, such as a meat sample.
[0436] In another aspect, the invention relates to a method for
detecting Streptococcus pneumoniae or parts thereof in a sample
comprising the step of analysing a sample by mass spectrometry to
evaluate the presence and/or quantity of one or more of the
polypeptides of SEQ ID NO:1-282, in particular SEQ ID NO:16 and/or
SEQ ID NO:20. In one embodiment, the sample, e.g. a blood sample,
is pre-treated to enrich for the polypeptide(s) to be detected.
Such a pre-treatment may include a size-fractionation of proteins
present in the sample.
[0437] The above methods can e.g. be used to diagnose Streptococcus
pneumoniae infections in an individual. In preferred embodiments of
the above methods, said indicator moiety does not pass through the
membrane of a Streptococcus pneumoniae cell. A preferred type of
said indicator moiety consists of or comprises an antibody, such as
an antibody of the invention as defined herein.
[0438] Those skilled in the art will understand that there are
numerous well known clinical diagnostic chemistry procedures in
which an indicator moiety can be used to form an binding reaction
product whose amount relates to the amount of the ligand, herein
Streptococcus pneumoniae or parts thereof, in a sample. Thus, while
exemplary assay methods are described herein, the invention is not
so limited.
[0439] The present invention also relates to a diagnostic system,
preferably in kit form, for assaying for the presence, and
preferably also the amount, of Streptococcus pneumoniae in a
biological sample. Methods for the preparation of diagnostic kits
have e.g. been described in U.S. Pat. No. 5,470,958 and references
therein.
[0440] The diagnostic system includes, in an amount sufficient to
perform at least one assay, an indicator moiety according to the
present invention, preferably as a separately packaged reagent, and
more preferably also instructions for use. Packaged refers to the
use of a solid matrix or material such as glass, plastic (e.g.,
polyethylene, polypropylene or polycarbonate), paper, foil and the
like capable of holding within fixed limits an indicator moiety of
the present invention. Thus, for example, a package can be a glass
vial used to contain milligram quantities of a contemplated
labelled indicator moiety preparation, or it can be a microtiter
plate well to which microgram quantities of a contemplated
indicator moiety has been operatively affixed, i.e., linked so as
to be capable of binding a ligand.
[0441] "Instructions for use" typically include a tangible
expression describing the reagent concentration or at least one
assay method parameter such as the relative amounts of reagent and
sample to be admixed, maintenance time periods for reagent-sample
admixtures, temperature, buffer conditions and the like.
[0442] In most embodiments, the diagnostic method and system of the
present invention include as a part of the Indicator moiety, a
label or indicating means capable of signalling the formation of a
binding reaction complex containing an indicator moiety complexed
with the preselected ligand (i.e. a polypeptide comprising any of
the sequences of SEQ ID NO:1-282 and/or a fragment thereof). Such
labels are themselves well-known in clinical diagnostic
chemistry.
[0443] The labelling means can be a fluorescent labelling agent
that chemically binds to antibodies or antigens without denaturing
them to form a fluorochrome (dye) that is a useful
immunofluorescent tracer. Suitable fluorescent labelling agents are
fluorochromes such as fluorescein isocyanate (FIC), fluorescein
isothiocyante (FITC), 5-dimethylamine-1-naphthalenesulfonyl
chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC),
lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC). Other
examples of suitable fluorescent materials include umbelliferone,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin and the like. A description of immunofluorescence
analysis techniques is found in DeLuca, "Immunofluorescence
Analysis", in Antibody As a Tool, Marchalonis, et al., eds., John
Wiley & Sons, Ltd., pp. 189-231 (1982).
[0444] Radioactive elements can be useful as labelling agents. An
exemplary radiolabeling agent is a radioactive element that
produces gamma ray emissions. Elements which themselves emit gamma
rays, such as .sup.124I, .sup.125I, .sup.128I, .sup.132I and
.sup.51Cr represent one class of gamma ray emission-producing
radioactive element indicating groups. Particularly preferred is
.sup.125I. Another group of useful labelling means are those
elements such as .sup.11C, .sup.18F, .sup.15O and .sup.13N which
themselves emit positrons, or beta emitters, such as .sup.111indium
of .sup.3H. Other suitable radioactive materials include .sup.131I
and .sup.35S.
[0445] Detection using antibodies can, in other embodiments, be
facilitated by coupling the antibody to another detectable
substance, such as an enzyme, a prosthetic group, a luminescent
materials, or a bioluminescent material. Examples of suitable
enzymes include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include Streptavidin/biotin and
avidin/biotin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin.
[0446] In preferred embodiments, the indicating group is an enzyme,
such as horseradish peroxidase (HRP) or glucose oxidase. In such
cases where the principal indicating group is an enzyme such as HRP
or glucose oxidase, additional reagents are required to visualise
the fact that a indicator-moiety/ligand complex (immunoreactant)
has formed. Such additional reagents for HRP include hydrogen
peroxide and an oxidation dye precursor such as diaminobenzidine.
An additional reagent useful with glucose oxidase is
2,2'-amino-di-(3-ethyl-benzthiazoline-G-sulfonic acid).
[0447] The linking of labels, i.e. labelling of polypeptides such
as antibodies, is well known in the art. For instance, proteins can
be labelled by metabolic incorporation of radioisotope-containing
amino acids provided as a component in the culture medium. See, for
example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The
techniques of protein conjugation or coupling through activated
functional groups are particularly applicable. See, for example,
Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978),
Rodwell et al. (1984) Biotech. 3:889-894, and U.S. Pat. No.
4,493,795.
[0448] Various diagnostic assays employing the above indicator
moieties can be set up to test samples for Streptococcus
pneumoniae. Exemplary assays are described in detail in Antibodies:
A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor
Laboratory Press, 1988. Representative examples of such assays
include: countercurrent immuno-electrophoresis (CIEP),
radioimmunoassays, radioimmuno-precipitations, enzyme-linked
immuno-sorbent assays (ELISA), Western blot assays, dot blot
assays, inhibition or competition assays, and sandwich assays,
immunostick (dipstick) assays, simultaneous immunoassays,
immunochromatographic assays, immunofiltration assays, latex bead
agglutination assays, immunofluorescent assays, biosensor assays,
and low-light detection assays (see e.g. also U.S. Pat. Nos.
4,376,110 and 4,486,530). An example of a suitable assay is an
assay wherein a sample, e.g. a serum sample, is separated by
electrophoresis and the polypeptide of interest, e.g. SEQ ID NO:16,
is subsequently detected by Western blotting.
[0449] In one embodiment, the diagnostic kits of the present
invention can be used in an "ELISA" format to detect the quantity
of a preselected ligand in a fluid sample. "ELISA" refers to an
enzyme-linked immunosorbent assay that employs an antibody or
antigen bound to a solid phase and an enzyme-antigen or
enzyme-antibody conjugate to detect and quantify the amount of an
antigen present in a sample and is readily applicable to the
present methods. Thus, in some embodiments, an indicator moiety of
the present invention can be affixed to a solid matrix to form a
solid support that comprises a package in the subject diagnostic
systems. A reagent is typically affixed to a solid matrix by
adsorption from an aqueous medium although other modes of
affixation applicable to polypeptides, such as antibodies, can be
used that are well known to those skilled in the art. Useful solid
matrices are also well known in the art. Such materials are water
insoluble and include the cross-linked dextran available under the
trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway,
N.J.); agarose; beads of polystyrene beads about 1 micron to about
5 millimetres in diameter available from Abbott Laboratories of
North Chicago, Ill.; polyvinyl chloride, polystyrene, cross-linked
polyacrylamide, nitrocellulose- or nylon-based webs such as sheets,
strips or paddles; or tubes, plates or the wells of a microtiter
plate such as those made from polystyrene or polyvinylchloride.
[0450] A further diagnostic method may utilise the multivalency of
an antibody composition of one embodiment of this invention to
cross-link ligands, thereby forming an aggregation of multiple
ligands and polypeptides, producing a precipitable aggregate. This
embodiment is comparable to the well known methods of immune
precipitation. This embodiment comprises the steps of admixing a
sample with a composition comprising an antibody of this invention
to form a binding admixture under binding conditions, followed by a
separation step to isolate the formed binding complexes. Typically,
isolation is accomplished by centrifugation or filtration to remove
the aggregate from the admixture. The presence of binding complexes
indicates the presence of the preselected ligand to be
detected.
Binding Partners and Inhibitors of Polypeptides of the
Invention
[0451] The surface-localisation of the polypeptides to which this
invention relates makes them highly suitable as targets for binding
partners, such as Inhibitors. Surface-located polypeptides of a
pathogenic microorganism often interact with the host organism.
Thus, any type of binding partner of a surface-located polypeptide
may interfere with host-pathogen interaction. Binding partners thus
often antagonise the pathogenicity (virulence) of a microorganism.
A binding partner may also be an inhibitor of the polypeptide it
binds.
[0452] Thus, in a further main aspect, the invention relates to
methods for the identification of binding partners of the
surface-located polypeptides set forth in SEQ ID NO:1-282. Such
methods may be biochemical or cell-based.
Biochemical Methods
[0453] In a main aspect, the invention relates to a method for
identifying a binding partner of a polypeptide selected from the
group consisting of SEQ ID NO:1-282, or a fragment thereof,
comprising the steps of [0454] a. providing a polypeptide selected
from the group consisting of SEQ ID NO:1-282 preferably selected
from the group consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID
NO:13, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:33,
most preferably SEQ ID NO:16, [0455] or [0456] a fragment thereof,
[0457] b. contacting said polypeptide or fragment with a putative
binding partner, and [0458] c. determining whether said putative
binding partner is capable of binding to said polypeptide or
fragment. In a preferred embodiment, said putative binding partner
is a host-derived molecule.
[0459] In further preferred embodiments of the method, the
polypeptide or fragment thereof is provided immobilised on a solid
support, such as e.g. a column or microtiter plate, and, after the
contacting step, it is determined whether or not the putative
binding partner has bound to the solid support. Immobilisation of
the polypeptide or fragment thereof may be through direct binding
to the solid support, or through indirect binding e.g. using a
specific antibody. In preferred embodiments, a washing step is
performed between the contacting step and the determination step,
in order to improve the specificity of detection. In further
preferred embodiments, the putative binding partner is complexed
with a detectable label. The putative partner may be labelled
before the contacting takes place. Alternatively, labelling may
also be performed after the contacting step. Furthermore, in some
embodiments of this method, immobilisation may be performed after
the polypeptide or fragment thereof has been bound to the binding
partner. In preferred embodiments, the method is a screening method
wherein the method is repeated for a plurality of putative binding
partners. Suitable methods to determine binding are well-known in
the art, and several of them have been referred to elsewhere
herein.
[0460] In another aspect, a host-derived binding partner of a
polypeptide selected from the group of SEQ ID NO:1-282, preferably
SEQ ID NO:16 may be identified as follows: purified host membranes
are electrophoretically separated, blotted over to a membrane and
incubated with the polypeptide of interest or fragment thereof.
Binding can then be detected using antibodies specific for the
polypeptide of interest or fragment thereof. The host binding
partner to which the polypeptide or fragment thereof has bound can
subsequently be identified by elution from the blot and subsequent
analysis by mass spectrometry, or by any other technique known in
the art.
[0461] If the binding partner of a surface-located polypeptide of a
pathogenic organism is a host-derived molecule, then such an
interaction between the surface-located polypeptide and the host
may be important for the virulence of the bacterium. Compounds that
interfere with the interaction of the surface-located polypeptide
and the host-derived binding partner may thus be suitable for
prevention or treatment of Streptococcus pneumoniae infections.
Accordingly, another method of the invention relates to a method of
identifying an inhibitor of the interaction of any of the
surface-located Streptococcus pneumoniae polypeptides of SEQ ID
NO:1-282 with a host-derived binding partner comprising the steps
of: [0462] a. providing any of the polypeptides of SEQ ID NO:1-282,
preferably selected from the group consisting of SEQ ID NO:16, SEQ
ID NO:10, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28
and SEQ ID NO:33, most preferably SEQ ID NO:16 [0463] or [0464] a
fragment thereof, [0465] b. providing a host-derived binding
partner of said polypeptide (identified as described above or by
any other method), [0466] c. contacting said polypeptide with said
host-derived binding partner in the absence of a putative inhibitor
of said interaction, [0467] d. contacting said polypeptide with
said host-derived binding partner in the presence of said putative
inhibitor, [0468] and [0469] e. determining whether the strength of
the binding of said polypeptide to said host-derived binding
partner resulting from step d. is reduced as compared to that
resulting from step c. In some embodiments, step c. and d. may be
performed in two different sample compartments. In other
embodiments, step d. may be performed by adding the putative
inhibitor to the mixture of step c. In preferred embodiments, the
method is repeated for a plurality of putative inhibitors.
[0470] Of particular interest are binding partners that inhibit an
activity of a surface-located polypeptide. Such activity may be
enzymatic activity, transport activity, or any type of other
biochemical or cellular activity, preferably enzymatic
activity.
[0471] Preferred host-derived binding partners are host
polypeptides and host lipids. Binding may e.g. be determined as
described by Szymanski and Armstrong (1996) Infect. Immun.
64:3467-3474.
[0472] In preferred embodiments of the above described biochemical
methods, the binding between the binding partner and the
surface-located polypeptide or fragment thereof has a dissociation
constant or Kd of less than 5.times.10.sup.-6M, such as less than
10.sup.-6M, e.g. less than 5.times.10.sup.-7M, such as less than
10.sup.-7M, e.g. less than 5.times.10.sup.-8M, such as less than
10.sup.-8M, e.g. less than 5.times.10.sup.-9M, such as less than
10.sup.-9M, e.g. less than 5.times.10.sup.-10M, such as less than
10.sup.-10 M, e.g. less than 5.times.10.sup.-11M, such as less than
10.sup.-11M, e.g. less than 5.times.10.sup.-12M, such as less than
10.sup.-12M. Dissociation constants can e.g. be determined by
surface plasmon resonance analysis.
Cell-Based Methods
[0473] Reducing the level of a surface-located polypeptide, by
deletion or disruption of the structural gene for it or by
down-regulating gene expression (see below), may affect a bacterial
cell. The cell may become more sensitive to cytotoxic compounds.
Especially for surface-located polypeptides, a reduction of their
level may affect the function of the cell's exterior parts, such as
the membrane or cell wall, in preventing compounds of entering the
cell. Thus, reduction of the level of an surface-located
polypeptide can make a cell more `permeable` for various
compounds.
[0474] Thus, an aspect of the present invention relates to a method
for identifying a compound with antibacterial activity against
Streptococcus pneumoniae comprising the steps of [0475] a.
providing a sensitised cell which has a reduced level of any of the
polypeptides of SEQ ID NO:1-282, preferably selected from the group
consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:33, most preferably
SEQ ID NO:16 [0476] and [0477] b. determining the sensitivity of
said cell to a putative antibacterial compound, for instance by a
growth assay.
[0478] Preferably, the method is a screening method wherein the
procedure is repeated for a plurality of putative antibacterial
compounds. Preferred putative antibacterial compounds are ones that
do not pass through the membrane of a wild-type Streptococcus
pneumoniae cell.
[0479] The rationale behind this approach is that a cell with a
lower level of the surface-located polypeptide will exhibit
increased sensitivity to cytotoxic compounds, allowing
identification of antibacterial compounds with low potency that are
missed when using wild-type cells for the assay. Compounds
identified by this method will be often need to be modified in
order to improve potency. This can be done by chemical
modification.
[0480] Inhibition of the activity of a surface-located polypeptide
may affect the viability (i.e. survival, growth and/or
proliferation) of the bacterium. Of particular interest is
inhibition of surface-located polypeptides that are essential for
viability of Streptococcus pneumoniae. Methods for testing
essentiality of a Streptococcus pneumoniae gene have been described
in the prior art, e.g. in Chan et al. (2002) J. Bacteriol
185:2051-2058 and Thanassi et al. (2002) Nucleic Acid Res.
30:3152-31-62. Inhibitors of essential surface-located polypeptides
may not need to enter the bacterial cell to be able to affect its
viability. Thus, generally fewer requirements are posed on the
structure of an inhibitor of an essential surface-located target
polypeptide than on an inhibitor of an intracellular target, to be
effective as an antibacterial agent.
[0481] Accordingly, the invention relates to a method for
identifying an inhibitor of a polypeptide selected from the group
consisting of SEQ ID NO:1-282, comprising the steps of [0482] a.
providing two cells which differ in the level of any of the
polypeptides of SEQ ID NO:1-282, preferably selected from the group
consisting of SEQ ID NO:16, SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:20, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:33, most preferably
SEQ ID NO:16 [0483] b. determining the sensitivity of said cells to
a putative inhibitor, for instance by a growth assay, and [0484] c.
determining whether said two cells are differently affected by the
presence of said putative inhibitor. Preferably, the method is
repeated for a plurality of putative inhibitors. Preferred
inhibitors are ones that do not pass through the membrane of a
Streptococcus pneumoniae cell.
[0485] The rationale behind this approach is that the viability of
a cell with a lower activity of the essential polypeptide will be
more affected by an inhibitor of the polypeptide than the viability
of the cell with a higher level. If the two cells are differently
affected, this is an indication that the inhibitor acts on the
target or at least in the same biochemical pathway.
[0486] In some embodiments of the method, the two cells with
different activity of the polypeptide of interest are a wild-type
cell (or other cell with wild-type activity of the gene of
interest) and a sensitised cell with a reduced activity of the
polypeptide of interest. In some embodiments, the different or
reduced level in the sensitised cell can be a different or reduced
expression level of the gene of interest (resulting in a different
or reduced copy number of the polypeptide). This can be
accomplished by putting the gene under control of a regulatable
promoter or by regulatable expression of an antisense RNA which
inhibits translation of an mRNA encoding the essential polypeptide.
In other embodiments, the different or reduced activity can be a
different or reduced activity of the polypeptide of interest, e.g.
due to a mutation, such as a temperature-sensitive mutation.
[0487] Suitable ways of generating sensitised bacterial cells and
of using these in screening for inhibitors have been described in
WO 02/077183. Sensitised cells may be obtained by growing a
conditional-expression Streptococcus pneumoniae mutant strain in
the presence of a concentration of inducer or repressor or other
conditions which provide a level of a gene product required for
bacterial viability such that the presence or absence of its
function becomes a rate-determining step for viability. The
sub-lethal expression of the target gene may be such that growth
inhibition is at least about 10%, such as at least about 25%, e.g.
at least about 50%, such as at least about 75%, e.g. at least 90%,
such as at least 95%.
[0488] In another embodiment of the cell-based assays of the
present invention, sensitised cells are obtained by reduction of
the level activity of a polypeptide required for bacterial
viability using a mutation, such as a temperature-sensitive
mutation, in the polypeptide. Growing such cells at an intermediate
temperature between the permissive and restrictive temperatures
produces cells with reduced activity of the gene product. It will
be appreciated that the above method may be performed with any
mutation which reduces but does not eliminate the activity or level
of the gene product which is required for bacterial viability. This
approach may also be combined with the conditional-expression
approach. In this combined approach, cells are created in which
there is a temperature-sensitive mutation in the gene of interest
and in which this gene is also conditionally-expressed.
[0489] When screening for inhibitors of an essential polypeptide,
growth inhibition can be measured by directly comparing the amount
of growth, measured by the optical density of the culture relative
to uninoculated growth medium, in an experimental sample with that
of a control sample. Alternative methods for assaying cell
proliferation include measuring green fluorescent protein (GFP)
reporter, construct emissions, various enzymatic activity assays,
and other methods well known in the art. Other parameters used to
measure viability include e.g. colony forming units. The above
method may be performed in solid phase, liquid phase, a combination
of the two preceding media, or in vivo. Multiple compounds may be
transferred to agar plates and simultaneously tested using
automated and semi-automated equipment. Cell-based assays of the
present invention are capable of detecting compounds exhibiting low
or moderate potency against the target molecule of interest because
such compounds are substantially more potent on sensitised cells
than on non-sensitised cells. The effect may be such that a test
compound may be two to several times more potent, e.g. at least 10
times more potent, such as at least 20 times more potent, e.g. at
least 50 times more potent, such as at least 100 times more potent,
e.g. at least 1000 times more potent, or even more than 1000 times
more potent when tested on the sensitised cells as compared to
non-sensitised cells.
[0490] A mutant Streptococcus pneumoniae strain that overexpresses
a surface-located polypeptide can also be used to identify a
compound that inhibits such a polypeptide. If the compound is
cytotoxic, overexpression of the target polypeptide can make cells
more resistant. Thus, the invention also relates to a method for
finding an inhibitor of any of the surface-located Streptococcus
pneumoniae polypeptides of SEQ ID NO:1-282 comprising the steps of
[0491] a. providing two cells which differ in the activity of any
of the surface-located Streptococcus pneumoniae polypeptides of SEQ
ID NO:1-282, preferably selected from the group consisting of SEQ
ID NO:16, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:26,
SEQ ID NO:28 and SEQ ID NO:33, most preferably SEQ ID NO:16 [0492]
wherein one cell contains a substantially wild-type copy number of
said polypeptide and the other cell contains higher than wild-type
copy number of said polypeptide, [0493] b. determining the
sensitivity of said cells to a putative inhibitor, for instance by
a growth assay, and [0494] c. determining whether or not said two
cells are differently affected by the presence of said putative
inhibitor. Overexpression may be achieved using strong promoters or
by introducing multiple copies of the structural gene for a
surface-located polypeptide. As also overexpression of polypeptides
that are not the cellular target of an inhibitor can make cells
resistance to an inhibitor, inhibition of the target polypeptide of
interest by a putative inhibitor will need to be verified by other
means, such as e.g. a biochemical assay.
[0495] In addition to inhibitors of a biochemical or other cellular
activity of a surface-located polypeptide, the cellular methods
described above can be used to identify compounds that reduce the
expression level of a target, and thereby its copy number, e.g. by
interfering with gene regulation.
[0496] In preferred embodiments of the any of the cell-based- or
biochemical methods for finding binding partners or inhibitors, the
method is repeated for a plurality of candidate compounds.
[0497] In a further aspect, the invention relates to the mutant
Streptococcus pneumoniae strains used in the cell-based methods
described herein, such as strains in which the gene encoding the
surface-located polypeptide is placed under the control of a
heterologous regulatable promoter, strains carrying
temperature-sensitive alleles of the surface-located polypeptides,
and strains overexpressing the surface-located polypeptides.
[0498] Other methods of interfering with bacterial growth by
targeting surface-located polypeptides, such as any of the
polypeptides of SEQ ID NO:1-282 include suppression of gene
expression using specific antisense molecules, such antisense RNA
or DNA, and using ribozyme molecules specific for mRNA encoding the
essential surface-located polypeptides.
Example 1
Strategy
[0499] The experimental steps in the project are as follows:
Surface proteins were isolated by either high pH elution or by
mutanolysin digestion. Isolated of surface proteins were identified
by three complementary mass spectrometric based strategies: 1) 2-D
SDS PAGE, 2) 1D SDS PAGE and 3) In-solution digest. All three
strategies include protein identification by mass spectrometry
analysis. The surface identified proteins are cloned into an E.
coli expression vector. The expressed recombinant proteins are
purified and used to immunise mice to verify immunogenicity of the
antigens. The immunised mice are used in challenge studies in which
the mice are challenged with Streptococcus pneumoniae and
protection against disease and/or death is monitored.
Mice:
[0500] Six-weeks-old BALB/c mice were housed under
specific-pathogen-free conditions and given sterile food and water
ad libitum.
Bacteria:
[0501] Escherichia coli Top10 (Invitrogen) was used as the host for
routine plasmid cloning. Recombinant proteins were expressed in E.
coli BL21/(DE3) (Invitrogen). E. coli were cultured in Luria broth
supplemented with antibiotics. Virulent S. pneumoniae strain D39
(serotype 2, purchased by Dr. M. Trombe, CICT, Toulouse, France)
was used for proteomics, challenge experiments and as a source of
genomic DNA for PCR amplification experiments. Clinical isolates of
S. pneumoniae, including 40 serotypes responsible for the majority
of pneumococcal infections were selected and purchased from the WHO
Collaborating Centre for Reference and Research on Pneumococci in
Copenhagen, Denmark. S. pneumoniae were routinely grown on blood
agar plates (Difco).
Isolation of S. pneumoniae Cell Envelope Fraction:
[0502] Mutanolysin digestion of pneumococcal cell wall. Bacteria
were grown overnight on blood agar plates, harvested into
phosphate-buffered saline (PBS) containing 20% sucrose and pelleted
by centrifugation at 6000 g for 10 minutes. The pellet was
resuspended in 0.5 ml of osmotic digestion buffer (20% sucrose in
20 mM Tris-HCl, pH 7.0, 10 mM MgCl.sub.2, protease inhibitor
cocktail and 100 U/ml mutanolysin (Sigma) per plate. Enzymatic
digestion was allowed to proceed for 1-2 h at 37.degree. C. The
intact protoplasts were removed by centrifugation at 7,000.times.g
for 15 min. The supernatant was collected, acetone precipitated and
analysed using mass spectrometry based techniques.
[0503] High pH elution of surface proteins. Bacteria were grown
overnight on blood agar plates, harvested into PBS containing 20%
sucrose and pelleted by centrifugation at 6000 g for 10 minutes.
The pellet was resuspended in PBS containing 20% sucrose and
centrifuged again as above. Then bacteria were resuspended in 2 ml
of 50 mM glycine-NaOH (pH 12) containing 20% sucrose per plate.
Alkali extraction of cell surface proteins was allowed to proceed
for 30 minutes at room temperature with gentle shaking. The
suspension was centrifuged at 15,000 g for 20 min, the supernatant
was collected, adjusted to pH 7 with 1 M HCl, acetone precipitated
and analysed by 1-D and 2-D gel electrophoresis.
Surface Protein Identification:
[0504] The complex mixture of proteins obtained after surface
extraction was analysed by three complementary strategies all based
on mass spectrometry: 2D gel based strategy, 1D gel based strategy
and In-solution digest strategy.
[0505] 2-D gel based strategy (2D-gel MALDI-TOF/TOF):
Two-dimensional gel electrophoresis was performed either on the
Ettan Dalt 2 system (Amersham Biosciences) or on the Novex NuPage
system (Invitrogen) according to the manual provided with the gel
system. In brief: First dimension runs were performed on either 7
cm or 24 cm pre-cast IPG strips (pH range 3-10 or 4-7) using the
Ettan IPGphor isoelectric focusing system (Amersham Biosciences)
according to the manufacturer's instructions. Isofocusing was
performed at the following conditions: 7 cm strips: 8000 Vh, 24 cm
strips: 52000 Vh. The second dimension was performed using pre-cast
12.5% gels (Amersham Biosciences) at 5 W per gel for 15 min then
total 170 W for 4-6 hours for 24 cm strips. The 7 cm strips were
run on the Novex NuPage system (Invitrogen) using pre-cast 4-12%
gels (Invitrogen) at 200 volts for 40 minutes. Gels were silver
stained according to a modified method described originally by
Mortz et al. (2001) Proteomics 1(11), 1359-1363, and spots for mass
spec analysis were picked using the Ettan Spot Picker from Amersham
according to the manufacturer instructions.
[0506] Specific protein spots were spot-picked, and placed in
Milli-Q water. These gel plugs were washed in 50 mM
NH.sub.4HCO.sub.3/50% ethanol and dehydrated by incubation in 96%
ethanol. Reduction and alkylation was performed by incubating in
reducing solution (10 mM DTT, 50 mM NH.sub.4HCO.sub.3) at
56.degree. C. followed by a room temperature incubation in
alkylation solution (55 mM iodoacetamide, 50 mM NH.sub.4HCO.sub.3)
in the dark. Two cycles of washing and dehydration were then
performed prior to the addition of 5 ul trypsin solution (12.5
ng/ul Promega trypsin in 50 mM NH.sub.4HCO.sub.3, 10%
Acetonitrile). Then an additional amount of sodium bicarbonate
solution was added and the digests were incubated overnight at
37.degree. C. Trifluoroacetic acid was added to the overnight
digest followed by incubation with shaking.
[0507] Parts of the extract were used in MALDI-TOF peptide mass
fingerprint and MALDI-TOF/TOF analysis (Ultraflex, Bruker
Daltonics, Germany) and the peak-lists were used in database
searching against a specific S. pneumoniae database. The Mascot
search program and scoring algorithm (Matrix Science, UK) was used
in database searching. Peptide mass tolerance was set to 60 ppm and
0.7 Da, respectively. Search parameters were adjusted to include
oxidation of Met, the addition of Carbamidomethyl groups to Cys,
and trypsin was allowed to miss one cleavage site per peptide.
[0508] 1D gel based strategy (GeLC-MS/MS): One-dimensional gel
electrophoresis was performed on a Novex NuPage system (Invitrogen)
according to the manual provided with the gel system. In brief we
used, size 8 cm.times.8 cm, 1 mm thick pre-cast 12% bis-tris gels
(Invitrogen) at 200 volts for 60 minutes in NuPage-MOPS-SDS running
buffer. Gels were silver stained according to a modified method
described originally by Mortz et al. (2001) Proteomics 1(11),
1359-1363. Whole lanes were cut out with razorblade in 0.5 cm gel
slices. The gel slices were digested as described under 2D gel
bases strategy, but the amount of trypsin was 20 ul trypsin
solution (12.5 ng/ul Promega trypsin in 50 mM NH.sub.4HCO.sub.3,
10% Acetonitrile).
[0509] The extracts was analysed on a LC-MS/MS (Waters Cap-LC and
Micromass Ultima TOF MS). Each extract was submitted to a 115
minutes LC-MS/MS analysis. The peak lists generated from the
fragmented peptides were used in database searching against a
specific Streptococcus pneumoniae database. The Mascot search
program and scoring algorithm (Matrix Science, UK) was used in
database searching. Peptide mass tolerance was set to 200 ppm and
0.4 Da for fragment ions. Search parameters were adjusted to
include oxidation of Met, the addition of Carbamidomethyl groups to
Cys, and trypsin was allowed to miss one cleavage site per
peptide.
[0510] In-solution based strategy (ISD-MS/MS): The protein mixture
was resuspended in 50 mM NH.sub.4HCO.sub.3, 10% Acetonitrile.
Trypsin solution (50 .mu.l) (12.5 ng/.mu.l Promega trypsin in 50 mM
NH.sub.4HCO.sub.3, 10% Acetonitrile) was added and the mixture was
incubated overnight at 37.degree. C. The digestion was stopped by
acidification with TFA (final conc. 1%) and analysed by LC-MS/MS as
described under 1D-based strategy. Database search was also
performed as described under 1D-based strategy.
Detection of Genes for Protein Vaccine Candidates in Different S.
pneumoniae Strains:
[0511] PCR amplification was used to demonstrate the presence of
genes encoding antigens listed in clinical isolates of S.
pneumoniae. For this purpose cells were grown on blood agar and
diluted in PBS. Genomic DNAs were prepared from 40 pneumococcal
strains by heating (95.degree. C. for 5 min) and aliquots were used
as templates for PCR amplification with Taq polymerase (Qiagen)
with gene specific primers. Amplification products were
electrophoresed through 1% agarose gels and visualized by staining
with ethidium bromide (0.5 .mu.g/ml).
Reverse Transcription Polymerase Chain Reaction (RT-PCR):
[0512] A BALB/C mouse was infected with S. pneumoniae D39 as
described below under Pneumococcal challenge. After 1 day of
infection this mouse was sacrificed and the spleen was extracted
and divided in two pieces. For isolation of intact total bacteria
RNA from tissue, one half the organ was frozen quickly in liquid
nitrogen and stored at -80.degree. C. before RNA isolation. The
other half of each organ was tested for bacteria using blood agar
plates before RNA isolation (data not shown). Total RNA was
isolated from animal tissue containing S. pneumoniae with the
Rneasy Kit (Qiagen, Hilden). First-strand cDNA synthesis was
performed with total RNA and the iScript Kit (Biorad). 1 .mu.l cDNA
were used for the subsequent PCR-step with gene specific
primers.
Production of Recombinant Vaccines (rec. vac.):
[0513] The production of recombinant vaccine was achieved by PCR
amplification of pneumococcal genes, with subsequent cloning and
expression of the genes in E. coli. Oligonucleotide primers used in
PCR amplification experiments were all purchased from MWG, Germany.
Pneumococcal genes used for protein expression were amplified from
genomic DNA of S. pneumoniae strain D39 by using the high-fidelity
thermostable DNA polymerase, Platinum Pfx (Life Technologies). The
coding sequence was amplified with primers containing the
start-codon but excluding the stop-codon of the open reading frame.
The coding sequences used for protein expression were cloned into
plasmid pET101 (invitrogen) using directional Topo cloning kit,
with E. coli Top10 as the bacterial host. A plasmid-encoded
C-terminal polyhistidine tag flanks each recombinant protein. For
recombinant protein expression, each recombinant pET101 plasmid was
subcloned into the E. coli strain BL21 (DE3). Recombinant protein
expression was initiated by induction with IPTG
(isopropyl-.beta.-D-thiogalactopyranoside), and proteins were
purified from the soluble fraction of recombinant E. coli lysates
by using metal affinity chromatography resin and buffers
(invitrogen), according to the manufacturer's instructions. Protein
concentrations were estimated by using the BCA test (Hercules,
Calif.). The recombinant proteins were dialyzed against PBS
(Millipore) and stored at -80.degree. C. Recombinant Proteins were
identified with MALDI MS technology after purification.
Western Blot Analysis:
[0514] Purified proteins were separated on one-dimensional (ca. 20
.mu.g protein) SDS-PAGE and transferred to PVDF membranes. The
immunological detection of immobilized proteins was performed after
manufactures instructions (invitrogen). Patient sera were kindly
provided by Dr. M. Trombe (CICT, Toulouse, France). We used a
dilution of 1:100 with single and pooled patient sera for
immunodetection. The dilution of the secondary antibody was
1:5000.
Pneumococcal Challenge of Actively Immunized Mice:
[0515] BALB/C mice were used in challenge experiments (10 mice per
group). For an antigen specific vaccination mice were primed with
25 .mu.g of each antigen containing the ALUM adjuvant (100 .mu.g)
on day zero. Animals were boosted with the same antigen
concentration on day 14 and day 28. We used untreated BALB/c mice
and mice treated with an unrelated cellulose-binding domain CBD
(sigma) as negative controls. All vaccines were administered
subcutaneously (s.c.). All mice were bled on days 0, 21 and 36 and
challenged on day 35 with S. pneumoniae D39. Individual sera from
each immunized mouse were tested for the presence of specific
antibodies prior to challenge. Virulent S. pneumoniae (D39) grown
on blood agar plates was prepared for challenge via the intra
peritoneal (i.p.) route in actively immunized mice or control
groups. For challenge infections, mice were injected i.p. with
approximately 10.sup.7 CFU per mouse of virulent S. pneumoniae
strain D39 suspended in PBS. The actual number of CFU administered
was determined retrospectively by plating serial dilutions of the
inocula on blood agar. The survival of mice was monitored for 7
days, at which time the experiments were terminated.
ELISA for Detection of Immunoglobulin G (IgG) Mouse:
[0516] Elisa assays were developed for detection of antigen
specific IgG in mouse sera at day 0, day 21 and day 35 after
vaccination. Different ELISA plates were coated with recombinant
vaccines (2 .mu.g) and whole cell lysates (2 .mu.g) of S.
pneumoniae D39. Two fold serial dilutions were made from mouse sera
as a primary antibody. The dilution of the secondary antibody (goat
anti mouse IgG, horse radish peroxidase conjugated) was 1:5000. OPD
substrate was used for the color development. Plates were read at
492 nm. Results of IgG status at day 21 and day 35 after
vaccination were compared with IgG status at day 0 of
vaccination.
Results:
[0517] We identified 282 different polypeptides in mutanolysin
created cell-surface fractions of S. pneumoniae. Sequences of the
identified polypeptides are given in FIG. 2. The methods that were
employed identifies polypeptides that are expressed at a relatively
high level. We used three different strategies for the detection of
isolated S. pneumoniae surface proteins: a) a 2-D gel based
strategy, b) a strategy with 1-D gels and LC_MS/MS and c) an
in-solution based MS strategy. We selected ORFs of identified
surface proteins for characterization. ORFs of genes were amplified
with PCR and cloned directional into pET101. Recombinant proteins
were expressed in E. coli as described in materials and methods.
Four recombinant proteins were selected for further studies (table
3):
TABLE-US-00004 AnrP number Group# (antigen#, short#) Description 1
230653 (1, 653) hydrolase, putative [Streptococcus pneumoniae TI;
ca. 32 kDa (rec. vac.) 2 516029 (2, 029) lipoate-protein ligase,
putative [Streptococcus pneumoniae TIGR4]; ca. 40 kDa (rec. vac.) 3
800607 (3, 607) ATP-dependent C1p protease, proteolytic subunit;
ca. 24 kDa (rec. vac.) 4 944060 (4, 060) autoinducer-2 production
protein; 21 kDa (rec. vac.)
[0518] A PCR investigation demonstrated that genes of each of these
4 selected proteins are presented in 40 different serotypes of S.
pneumoniae (table 4).
TABLE-US-00005 TABLE 4 PCR with different S. pneumoniae serotypes
and specific primers for following genes (+ = detected; - = not
detected) Serotype AnrP230653 AnrP516029 AnrP800607 AnrP944060 1 +
+ + + 2 + + + + 3 + + + + 4 + + + + 5 + + + + 6a + + + + 6b + + + +
7f + + + + 7c + + + + 8 + + + + 9n + + + + 9v + + + + 9a + + + +
10a + + + + 10b + + + + 11a + + + + 12f + + + + 13 + + + + 14 + + +
+ 15b + + + + 15c + + + + 16f + + + + 17f + + + + 18b + + + + 18c +
+ + + 19a + + + + 19f + + + + 20 + + + + 22f + + + + 23a + + + +
23f + + + + 28f + + + + 31 + + + + 31f + + + + 32 + + + + 33f + + +
+ 34 + + + + 35b + + + + 35f + + + + 38 + + + +
[0519] For identification of transcripts of these 4 genes we made a
RT-PCR analysis. A BALB/C mouse was infected with S. pneumoniae and
total RNA was isolated from spleen after day 1 of infection.
RT-PCRs with RNAs from spleen demonstrated that the selected genes
are expressed in S. pneumoniae after animal infection (FIG. 3). In
addition Western Blots (WB) with patient sera were made for testing
the immunogenicity of proteins listed in table 3. Furthermore 3
additional proteins (144, AnrP454144 (14 kDa rec. vac.); 487,
AnrP98487 (32 kDa); 646; AnrP373646 (25 kDa)) were also tested for
immunogenicity with Western Blot. We detected recombinant vaccines
029, 060, 607, 646 and 653 in immunoblots (WBs) with sera isolated
from a single patient (FIG. 4) or pooled from different patients
(data not shown). In WBs unspecific signals or no signals were
detected for purified proteins 144 and 487, however, lack of a
signal does not exclude that these two proteins may be suitable as
vaccines.
[0520] We vaccinated mice with proteins in table 3 as a protein
vaccine and tested the protection efficiency against S. pneumoniae.
Antigens were prepared with alum and injected subcutaneously into
BALB/C mice at three time points (day 0, 14, 28). For negative
controls, mice were left untreated (group 5) or treated with 100 kg
alum (group 6) or with an unrelated protein (group 7). We tested
the immunogenicity of each protein at day 0, day 21 and day 35 of
vaccination with an ELISA assay. Mice produced immunoglobulin (IgG)
against antigens 029, 060, 607, and 653 (FIG. 5). No immune
response against pneumococcal antigens was detected as expected in
animals of groups 5, 6 and 7. For the bacterial challenge, each
mouse was infected with S. pneumoniae D39 (1 CFU per mouse) at day
35 of vaccination. Mice derived from two groups, vaccinated with
proteins 029 and 607 demonstrated a lower mortality and a lower
CFU-titre after infection with this S. pneumoniae strain (FIG. 6,
FIG. 7). Proteins 060 and 653 also showed a trend towards lower
CFU-titre (FIG. 6).
[0521] In order to investigate variations in sequence between
different strains of Streptococcus pneumoniae, the sequences of
antigens 029 and 607 were partially determined from serotypes 15b,
15c, and 35f, and from Streptococcus pneumoniae strain D39. These
sequences were compared with database sequences of 029 and 607 from
type 4 (TIGR) and R6 (Sanger Center). The region of 029 from amino
acid position 1 to amino acid position 315 showed more than 98%
sequence identity on amino acid level between the six strains. For
607, more than 98% sequence identity on amino acid level between
the six strains was found in the region from position 20 to
position 190. These data indicate that 029 and 607 are
well-conserved across different strains.
[0522] The structure of antigen 029 (SEQ ID NO:16) (putative
lipoate protein ligase) has been determined and is accessible under
accession number 1VQZ in the PDB (Protein Data Bank) and HSSP
databases of EBI, the European Bioinformatics Institute.
Surface-exposed regions were predicted by identification of amino
acids with high water accessibility (ACC), which at the same time
should have a low variability (VAR) in sequence (see table below).
The amino acid stretches of different areas separated by several
amino acids are adjacent in the 3D structure and are therefore
paired together.
Amino Acids Exposed on the Surface:
TABLE-US-00006 [0523] Amino acid No. Amino acid ACC VAR 127 I 7 21
128 D 139 26 129 G 36 18 155 D 78 20 156 L 90 24 157 S 72 35 158 V
9 49 159 L 53 14 160 A 83 42 166 S 15 37 167 K 102 35 168 D 59 37
169 K 74 7 170 F 34 22 171 E 92 41 172 S 86 26 173 K 39 13 174 G 64
10 175 V 78 11 176 K 96 28 177 S 77 0 178 V 100 11 179 R 102 20 180
A 86 32 185 I 0 14 186 I 44 48 187 N 110 32 188 E 51 46 189 L 18 18
190 P 102 42 191 K 70 36 195 V 1 43 196 E 84 26 197 K 112 39 198 F
0 22 199 R 37 43 200 D 72 40 201 L 33 42 202 L 0 25 203 L 24 38 204
E 116 46 207 K 65 51 208 K 102 41 209 E 81 50 210 Y 42 50 211 P 107
46 155-160 + 185-191 [DLSVLA (SEQ ID NO:289)/IINELPK (SEQ ID
NO:290)] 127-128 + 166-180 [IDG/SKDKFESKGVKSVRA (SEQ ID NO:291)]
195-204 + 207-211 [VEKFRDLLLE (SEQ ID NO:292)/KKEYP (SEQ ID
NO:293)]
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090202528A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090202528A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References